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Endocrinology 146(10):4234 – 4249
Copyright © 2005 by The Endocrine Society
doi: 10.1210/en.2005-0372
Cholesterol and Steroid Synthesizing Smooth
Endoplasmic Reticulum of Adrenocortical Cells Contains
High Levels of Proteins Associated with the
Translocation Channel
Virginia H. Black, Archana Sanjay, Klaus van Leyen, Brett Lauring, and Gert Kreibich
Department of Cell Biology and Kaplan Cancer Center (V.H.B., A.S., G.K.), New York University School of Medicine, New
York, New York 10016; Cellular Biochemistry and Biophysics Program (K.v.L.), Memorial Sloan-Kettering Cancer Center,
New York, New York 10021; and Department of Pathology (B.L.), Columbia University, New York, New York 10027
Steroid-secreting cells are characterized by abundant smooth
endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. Yet they have
relatively little morphologically identifiable rough endoplasmic reticulum, presumably required for synthesis and maintenance of the smooth membranes. In this study, we demonstrate that adrenal smooth microsomal subfractions enriched
in smooth endoplasmic reticulum membranes contain high
levels of translocation apparatus and oligosaccharyltransferase complex proteins, previously thought confined to
rough endoplasmic reticulum. We further demonstrate that
these smooth microsomal subfractions are capable of effect-
S
TEROID-SECRETING CELLS are characterized by abundant smooth endoplasmic reticulum (SER). These cells
synthesize cholesterol as a precursor for steroid hormones or
take up this substrate from plasma lipoproteins. The ratio of
synthesis to uptake is dependent on the species, cell type, and
functional state (see Ref. 1 for recent review). Many of the
enzymes for sterol and steroid synthesis are localized in the
smooth-surfaced endoplasmic reticulum (2, 3). This organelle is particularly prominent in cells of the inner zones
of the adrenal and fluctuates in amount and configuration in
First Published Online June 9, 2005
Abbreviations: BiP, Ig heavy chain-binding protein; bw, body weight;
CTF, C-terminal fragment; CYP, cytochrome P450; DAB, 3,3⬘-diaminobenzidine tetrahydrochloride; DAD, defender against apoptotic death;
ECL, enhanced chemiluminescence; EndoH, endoglycosidase H; ER,
endoplasmic reticulum; ERAD, ER-associated degradation; GRP, glucose regulated protein; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme
A reductase; 3␤HSD, 3␤-hydroxysteroid dehydrogenase; Hsp, heat
shock protein; 3MC, 3-methylcholanthrene; op-156, the first 156 codons
of bovine opsin; OST, oligosaccharyl-transferase; OTP, octanoyl tripeptide; PB, phenobarbital; PKRM, puromycin/KOAc washed microsomes;
86pPL, 86-amino-acid preprolactin; RAMP4, ribosome-associated membrane protein 4; RER, rough endoplasmic reticulum; RI and RII, ribophorins I and II; RNC, ribosome-nascent chain complex; SDS, sodium
dodecyl sulfate; SER, smooth endoplasmic reticulum; SP, signal peptide;
SPC, signal peptide cleavage protein, signal peptidase; SR, SRP receptor;
SRP, signal recognition particle; TCA, trichloroacetic acid; TLC, thinlayer chromatography; TM, transmembrane; TRAP, translocon-associated protein.
Endocrinology is published monthly by The Endocrine Society (http://
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endocrine community.
ing cotranslational translocation, signal peptide cleavage,
and N-glycosylation of newly synthesized polypeptides. This
shifts the paradigm for distinction between smooth and rough
endoplasmic reticulum. Confocal microscopy revealed the
proteins to be distributed throughout the abundant tubular
endoplasmic reticulum in these cells, which is predominantly
smooth surfaced. We hypothesize that the broadly distributed
translocon and oligosaccharyltransferase proteins participate in local synthesis and/or quality control of membrane
proteins involved in cholesterol and steroid metabolism in a
sterol-dependent and hormonally regulated manner. (Endocrinology 146: 4234 – 4249, 2005)
response to hormonal stimulation and sterol levels (4 –7). It
may assume the form of random tubules, arrays of fenestrated cisternae, or crystalloid configurations (4) (Fig. 1). The
more complex forms also occur in hepatocytes and cultured
cells in which 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), a key enzyme in cholesterol synthesis, is
overexpressed as well as in cultured cells overexpressing
other proteins characteristic of the SER, e.g. cytochromes
P450 (8). HMGR has a high turnover rate (9), and its levels
in adrenocortical cells are regulated in a sterol and ACTHdependent manner (10, 11). Changes in SER volume and
enzyme content with functional state indicate that there must
be fluctuations in synthesis and degradation of HMGR and
other enzymes in the cholesterol and steroid biosynthetic
pathway. Yet morphologically identifiable rough endoplasmic reticulum (RER), presumably required for these functions, is sparse in these cells (less than 0.3% of the cytoplasmic
volume) (6). Bound ribosomes occur on short endoplasmic
reticulum (ER) cisternae and in patches scattered along predominantly smooth-surfaced, randomly arranged tubules or
at the periphery of smooth cisternal and crystalloid arrays
(Figs. 1 and 2, arrows). Although the RER increases 2- to 4-fold
after ACTH treatment, this morphological appearance does
not change significantly (6). Cisternae densely covered with
ribosomes are rarely seen.
In contrast, cells specialized for production of secreted
proteins, such as those of the pancreas and liver, possess
prominent parallel arrays of RER cisternae, densely studded
with ribosomes (Fig. 1). Using microsomes prepared from
these cells, a large number of proteins have been identified
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Black et al. • Translocon Proteins in Adrenal SER
Endocrinology, October 2005, 146(10):4234 – 4249
4235
FIG. 1. Comparison of the ER in protein-secreting vs. steroid-secreting cells and the subcellular fractions derived from them. As shown on the
left, in the embryo and many cultured cells, the ER bears scattered patches of ribosomes. When protein-secreting cells differentiate, they acquire
ER characterized by arrays of ribosome-studded cisternae, the RER, which interconnects with tubular elements, lacking ribosomes, the SER.
In steroid-secreting cells, the ER becomes predominantly smooth surfaced, forming tubules or more complex arrays of fenestrated cisternae
and hexagonally packed tubules. Ribosomes are found on short cisternae and tubules, but cisternae densely covered with ribosomes are seldom
seen. Upon subcellular fractionation of both cell types, as indicated in the center panel, regions of the ER with bound ribosomes will be isolated
as rough microsomes and regions lacking ribosomes will be isolated as smooth microsomes. Key components of the ER membrane are depicted
diagramatically on the right. The components of the translocation apparatus or translocon and OST are aligned with the rough microsomal
fraction, in which they are localized in fractions obtained from protein-secreting cells, such as pancreas and liver. In steroid-secreting cells,
however, as shown in this paper, both translocon and OST complex proteins are also found in high concentrations in the smooth microsomal
fraction, which is enriched in enzymes of sterol and steroid metabolism.
that are involved in targeting, translocation and processing
of proteins synthesized on membrane bound ribosomes (12–
15) (for summary see Table 1). Some of these components
have been shown to be largely confined to rough microsomes
derived from the RER of protein-secreting cells: signal recognition particle (SRP) receptor (SR) (58), Sec61␣ (33), subunits of the translocon-associated protein (TRAP) complex
(59) as well as ribophorins I and II (RI and RII) (60 – 63).
In this study, to better understand the functional dynamics of the adrenal ER, we analyzed the levels of key
elements of the translocation apparatus and associated
proteins involved in processing of newly synthesized
polypeptides in microsomal subfractions obtained from
adrenals in comparison with microsomal subfractions prepared from liver and pancreas. We present the surprising
finding that many of the proteins involved in translocation
and processing of ER-targeted proteins, including those
considered as classical RER markers, e.g. subunits of the
Sec61 and oligosaccaryltransferase (OST) complexes, are
very abundant in adrenal smooth microsomes. Confocal
microscopy revealed that their distribution in situ was
similar to that of steroidogenic enzymes, which are localized predominantly in the SER. This observation shifts the
paradigm for the distinction between RER and SER, at
least for these cells, and raises questions about the function
of these complexes in the adrenal SER. We demonstrate
that these complexes are capable of their respective functions of ribosome binding and cotranslational translocation, signal peptide cleavage, and N-glycosylation of
newly synthesized peptides in this setting. We hypothesize that these proteins take part in regulating levels of SER
membrane components involved in the metabolism of cho-
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Endocrinology, October 2005, 146(10):4234 – 4249
Black et al. • Translocon Proteins in Adrenal SER
FIG. 2. The abundant SER in steroid-secreting cells facilitates preparation of very clean smooth microsomal subfractions. A, Electron micrograph of a cell from the guinea pig inner adrenal cortex, illustrating the abundant SER in these cells. RER is largely confined to short cisternae
or small patches of ribosomes scattered on the predominantly tubular ER (arrowheads). Bar, 1.0 ␮m. ⫻ 20,700. B, Electron micrograph of rough
microsomes obtained from this tissue. They are not as densely covered with ribosomes as corresponding fractions prepared from protein-secreting
cells such as pancreas or liver. Bar, 0.5 ␮m. ⫻ 40,000. C, Electron micrograph of smooth microsomes obtained from this same tissue. Few, if
any, ribosomes are visible on the surface of these microsomes. Bar, 0.5 ␮m. ⫻ 40,000.
lesterol and steroids in an ACTH- and sterol-dependent
manner.
Materials and Methods
Animals and treatment
English short-haired Hartley strain guinea pigs [600 – 800 g body
weight (bw)] and Sprague Dawley rats (250 g bw) were obtained from
Camm Research Laboratories (Camden, NJ) and Jackson Laboratories
(Bar Harbor, ME), respectively. They were maintained on standard
laboratory chow ad libitum in a controlled lighting environment (lights
on 0600 h; lights off 1900 h). Experimental protocols and animal care
were reviewed and approved by the Institutional Animal Care and Use
Committee. In three experiments, rats were injected with sodium phenobarbital (PB; 100 mg/kg, ip) or the saline vehicle alone for 4 d before
being killed. Guinea pigs were treated with 3-methylcholanthrene
(3MC), as previously described (64). Rats were killed by decapitation
before tissue removal. Guinea pigs were anesthetized with sodium pentobarbital (60 mg/kg bw, Nembutal, Abbot Laboratories, North Chicago, IL) before tissue removal and being killed. Tissues were removed,
placed on ice, and weighed immediately.
Cell fractionation
Microsomal subfractions from guinea pig adrenals and liver as well
as the adrenals of several other species obtained from Pel-Freez (Rogers,
AR) were prepared as previously described (65, 66). Liver microsomes
Black et al. • Translocon Proteins in Adrenal SER
Endocrinology, October 2005, 146(10):4234 – 4249
4237
TABLE 1. Major proteins of translocation apparatus and OST complex
Protein
Approximate
size (kDa)
Membrane orientation
Translocation apparatus
Sec61 complex
Sec61␣
40
Spans membrane 10
times
Sec61␤
13
Single spanning with
lumenal C-terminal tail
SR
SR␣
72
TRAM
Peripheral membrane
protein
Multispanning
glycoprotein with
cytosolic C-terminal tail
SERP1/RAMP4
TRAP
␣TRAP
34
Single spanning
N-glycosylated
Sec63 complex
Sec62
62
Sec63
98
Spans membrane twice,
has long cytosolic C
terminus
Spans membrane three
times, has long cytosolic
C terminus
Associated enzymes
SPC
SPC12
12
Spans membrane twice
OST complex
RI
68
RII
66
Ost48
48
DAD1
12.5
STT3A, STT3B
Ost3p Homologs,
N33-1, 2, IAP
Molecular chaperones
BiP
⬃65, ⬃80
32–35
78
Single spanning type 1,
bulk in lumen,
glycoprotein
Single spanning type 1,
bulk in lumen,
glycoprotein
Single spanning type 1,
large lumenal domain
non-glycosylated
Spans membrane twice
Span membrane 13 times,
glycosylated
Span membrane four
times
Lumenal protein
Grp94
94
Lumenal protein
Calnexin
60
Type I transmembrane
protein
Function
Involved in translocation of proteins into and out of the ER,
translocation into ER may be co- or posttranslational
Heterotrimeric complex of Sec61␣, Sec61␤, and Sec61␥, forms core of
the translocation apparatus, the translocon
Lines hydrophilic translocation channel, possesses ribosomal binding
site, interacts with SRP and its receptor, resulting in release of
nascent chain from SRP into translocon channel
Interacts with Sec61 subunits and 25-kDa subunit of signal
peptidase, capable of binding ribosomes, may be involved in
integration of newly synthesized membrane proteins into the lipid
bilayer
SRP receptor, a heterodimer consisting of an ␣ (72 kDa) and ␤ (30
kDa) subunit, both GTPases. The ␤-subunit coordinates signal
sequence release from SRP with ribosome binding to the translocon
Subunit of the signal recognition particle receptor, SR␣ is bound to
the cytosolic face of the ER by SR␤. It interacts with SRP54, also a
GTPase. Regulation of these two GTPases is reciprocal, ensuring
that each engages ligands before interacting with the other.
The translocating-chain associating membrane protein is required for
the translocation of some, but not all membrane proteins
SERP1/RAMP4 interacts with Sec61␣, Sec61␤, and calnexin. It
appears to suppress degradation of newly synthesized membrane
proteins in ER stressed cells and to facilitate their glycosylation
once the stress is removed.
Translocation apparatus associated protein, a heterotetrameric
complex found in association with native ribosome-channel
complexes. Two of the subunits are glycosylated (␣, ␤); the other
two are not. Facilitates translocation of specific substrates,
particularly those lacking a strong signal sequence.
Subunit of TRAP found in association with native ribosome-channel
complexes
Heterotetrameric complex (Sec62, Sec63, Sec71, and Sec72) that
associates with Sec61 complex to form apparatus involved in
posttranslational translocation in yeast. Sec62 and Sec63 are
essential and mammalian homologs have been found. Functions of
Sec71 and Sec72 are not known.
May provide binding site for signal sequence of nascent polypeptide
chain
Refs.
Reviewed in
Refs. 12–15
16–19
16, 20
21, 22
23–26
16–18, 27
17, 28
29, 31
30
32–34
35, 36
Lumenal J-like domain provides binding site for BiP, which acts as
an ATPase, providing energy for movement of nascent chain into
ER lumen by rachet type action
34, 37
Removes cleavable signal sequences; composed of five/six known
subunits, interacts with Sec61␤, may be transiently recruited to
the translocation site; may interact with OST complex to ensure
that signal peptide cleavage occurs before glycosylation.
Subunit of signal peptidase; cytoplasmic orientation of this subunit
suggests function in processes other than enzymatic cleavage.
Oligomeric complex involved in N-glycosylation of newly synthesized
polypeptides, transfers preassembled sugar chains to polypeptide
from dolichol phosphate carrier, exists in several isoforms
RI bears a putative dolichol recognition sequence, facilitates Nglycosylation
20, 38, 39
40
41–43
41
RII complexes with the lumenal domain of OST48 helping to retain
OST 48 in ER
44
Together with RI and STT3 forms catalytically active complex,
association with RI and RII affects ER retention of OST48
45, 46
Stabilizes the OST complex, interacts with several regulatory
proteins
Determine kinetic properties of OST isoforms, responsible for protein
substrate recognition and catalysis of N-linked glycosylation
Contain thioredoxin domains, may be less tightly associated with
OST complex, only N33-1 co-sediments with OST activity
Involved in folding of newly synthesized polypeptides
Belongs to heat shock protein family Hsp70. BiP seals the lumenal
side of the translocon and participates in posttranslational
translocation. It binds unfolded regions of nascent proteins and
directs folding. It recognizes improperly folded proteins and targets
them for degradation. It is a calcium binding protein and binds to
lumenal domains of ER stress inducers.
Belongs to heat shock protein family Hsp90, binds to selected
substrates
Lectin that binds monoglucosylated oligosaccharides, can also bind
nonglycosylated proteins, associates with Erp57, a thiol protein
oxidoreductase and isomerase, facilitating disulphide bond
formation
42, 47–49
SERP, Stress-associated endoplasmic reticulum protein; IAP, implantation-associated protein; Hsp, heat shock protein.
43
43
50, 51
37, 52–54
55
56, 57
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Endocrinology, October 2005, 146(10):4234 – 4249
were prepared from rats fasted for 18 h using this method, a modification
of that described by Adelman et al. (67), as well as by the modification
of Adelman’s method described by Kruppa and Sabatini (68). Dog pancreas rough microsomes were prepared as previously described (61). In
some cases, ribosomes were removed from the microsomal membranes
after subfractionation, using high salt treatment and puromycin, as
described previously (69). All fractions were stored at ⫺70 C.
Antibodies
The proteins associated with protein translocation and processing that
were investigated as a part of this study are listed in Table 1. Antibodies
directed against RI and RII, OST48, Ig heavy chain-binding protein (BiP)
[glucose regulated protein (GRP)78], and ␣TRAP were as previously described (47, 70). Dr. Andrei Nikonov made the antipeptide antibody to
defender against apoptotic death (DAD)1 (amino acids 76 –91) in the laboratory of one of the investigators (G.K.). The antibody to the SR, raised
against the 16 C-terminal amino acids of the ␣-subunit, was also made in
the laboratory (of G.K.). Antibodies for Sec61␣ and Sec61␤ were raised in
the laboratory of Dr. Martin Wiedmann (Memorial Sloan-Kettering Cancer
Center, New York, NY) and affinity purified by Dr. Robert Levy while a
student in one of the authors’ laboratories (G.K.). Antibodies against Sec62
and Sec63 were received from Dr. R. Zimmermann (Universität des Saarlandes, Sarbrucken, Germany). Antibody against GRP94 was obtained from
Dr. Christopher Nicchitta (Duke University, Durham, NC). Antibody for
signal peptidase SPC12 was from Dr. Enno Hartmann (University of Lubeck, Lubeck, Germany) (40). Antibody against the ribosomal S3 protein
was obtained from Dr. Vicky Richon and was made by Dr. Xianbo Zhou
in the laboratory of Drs. Paul Marks and Richard Rifkind (Memorial SloanKettering Cancer Center). Antibodies to enzymes involved in sterol and
steroid synthesis, HMGR, cytochromes P450 17␣-hydroxylase (CYP17), and
3␤-hydroxysteroid dehydrogenase (3␤HSD) as well as cytochromes P450
involved in xenobiotic metabolism, CYP1A and CYP2B, were as described
previously (11, 64, 66, 71, 72). All of the antibodies were raised in rabbits,
most against purified proteins, unless specified as antipeptides. All recognized proteins of the appropriate size in Western blots and most showed
minimal cross-reactivity with other proteins under the conditions
employed.
Immunoblotting
Microsomal proteins were separated by electrophoresis on 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred electrophoretically to nitrocellulose sheets, as previously described (66, 71).
Smaller proteins, DAD1 and Sec61␤, were separated electrophoretically
on 20% SDS-polyacrylamide gels in a Tris-tricine buffer system, according to published protocols (73). In general, 12 ␮g of protein were used
for each sample. However, protein concentrations were adjusted for
optimal visualization in some cases, as indicated in the figure legends.
Blots were stained with Ponceau S (Sigma Diagnostics, St. Louis, MO)
to visualize corun standards and check protein loading and transfer, as
previously described (66, 71). Tween 20 and/or 5% dry milk in PBS were
used as blocking agents. Peroxidase-conjugated secondary antibodies
(Capell, Durham, NC) were visualized using 3,3⬘-diaminobenzidine tetrahydrochloride (DAB; Polysciences, Warrington PA) or the enhanced
chemiluminescence (ECL) Western blotting analysis system (Amersham
Life Science, Buckinghamshire, UK). Once optimal conditions for each
antibody had been established, subsequent blots were cut into pieces so
that immunoreactions for proteins of differing sizes could be performed
on each set of samples in overlapping combinations, e.g. calnexin, RI or
RII, and Sec61␣; GRP94, BiP, RI or RII, CYPs, and Sec61␣ or ␣TRAP. This
permitted a more accurate assessment of the relative distribution of
multiple proteins among the microsomal subfractions. Blots for each
protein were run three to four times. Quantitation of immunoblots was
performed using NIH Image.
Immunocytochemistry
Isolated adrenocortical cells were prepared and grown on coverslips,
as previously described (74). After 3–5 d in culture, in the presence or
absence of ACTH (100 mU/ml), the cells were rinsed briefly with PBS
and fixed for 10 min on ice with methanol that had been stored at ⫺20
C. Subsequent incubations were carried out at room temperature in 1%
Black et al. • Translocon Proteins in Adrenal SER
milk in PBS [blocking, 1 h; primary antibody (1:200), 1–5 h; three rinses;
secondary antibody (fluorescein-conjugated AffiniPure F(ab⬘)2 fragment donkey antirabbit IgG (H⫹L), Jackson ImmunoResearch Laboratories Inc, West Grove PA)(1:125), 1 h], followed by several rinses in plain
PBS. Coverslips were adhered to glass slides with Citiflour mounting
medium (Citifluor mountant medium #0, Ted Pella, Redding, CA). Corun controls were incubated in the secondary antibody alone. Confocal
microscopy was performed with a LSM510 laser scanning microscope
(Zeiss, Göttinger, Germany).
Analysis of OST activity
The octanoyl tripeptide (OTP), N-octanoyl-Asn-Tyr-Thr-amide, which
contains the acceptor sequence for N-glycosylation, was received from Dr.
Felix Wieland (University of Heidelberg, Heidelberg, Germany). [125I]OTP
was prepared and used as a glycosyl acceptor to assay oligosaccharyltransferase activity, as previously described (75–77). After incubation with
microsomal protein (30 –300 ␮g) for 1 h at 37 C in the presence and absence
of the glucosidase inhibitor, castanospermine (50 ␮m), the glycosylated
tripeptide was bound to a conA Sepharose column and eluted with buffer
containing methyl-␣-d-mannopyranoside and Triton X-100. Activity in the
eluate from the conA Sepharose column is expressed as counts per minute
per microgram microsomal protein used in the assay.
Analysis of glycotripeptides by thin layer chromatography (TLC) was
performed as previously described (75–77). To further define the pattern of
glycosylation, the eluted samples were subjected to enzymatic digestion
with ␣-mannosidase and Endo H (77). Aliquots containing approximately
equal counts per minute from each sample were incubated with Jack bean
␣-mannosidase (6 U/ml; Glyko, Inc., Novato, CA) or Endo H (Roche,
Mannheim, Germany) in the 1⫻ buffer supplied with the ␣-mannosidase,
as described in the accompanying literature. The total incubation mixture
was 5–9 ␮l. After incubation at 37 C, overnight (⬃16 h), the reaction mixture
was spotted on TLC plates, without further processing and the components
separated in the solvent system described above.
Ribosome targeting and binding
For the targeting assay, truncated mRNA lacking a stop codon encoding the first 86 amino acids of preprolactin (86pPL) was translated
in a rabbit reticulocyte lysate supplemented with [35]S-Met and trifluoromethyldiazirinobenzoic acid-modified lys-tRNA as previously described (78) to create nascent chains modified for photocross-linking.
After translation, cycloheximide was added to 0.5 mm and microsomes
were added. After incubation for 2 min at room temperature and 10 min
on ice, samples were irradiated to induce cross-linking. The samples
were trichloroacetic acid (TCA) precipitated, resuspended in Laemmli
sample buffer, and electrophoresed through 12% gels. The 62-kDa photoadduct representing the 86pPL nascent chain cross-linked to SRP (54
kDa) was visualized by fluorography.
For the binding assay, 86pPL mRNA was translated in a wheat germ
lysate supplemented with [35]S-Met and high salt extracted ribosome/
nascent chain complexes (RNCs) were prepared and isolated as previously described (79). RNCs (5 ␮l) were incubated with puromycin/
KOAc washed dog pancreas microsomes (PKRMs, 10 mg/ml or 1 eq/␮l)
or adrenal smooth microsomes (12 mg/ml) for 2 min at room temperature and 10 min on ice and bound RNCs separated from unbound RNCs
by flotation in discontinuous sucrose gradients, as previously described
(79). The top gradient fractions containing membranes were collected
and analyzed for RNC content by autoradiography after SDS-PAGE.
Assay of cotranslational translocation and processing
Two truncated mRNAs were prepared. The first, used for assay of signal
peptide cleavage, encoded the C-terminal fragment (CTF; 99 amino acids)
of amyloid precursor protein with bovine pPL signal peptide (SP) sequence
added at the 5⬘ end. The second, used for assay of N-glycosylation, was
derived from plasmid pSF1, received from Dr. S. M. Simon (Rockefeller
University, New York, NY). pSF1 contained the complete cDNA of opsin
inserted in the Sac1 site of an SP6/4 vector (80). It was linearized with BsaH1
before runoff transcription with SP6 RNA polymerase (SP6 Cap-Scribe;
Roche Molecular Biochemicals, Mannheim, Germany) to prepare truncated
mRNA encoding the first 156 codons of bovine opsin (op-156). This region
includes the first three transmembrane-spanning segments and a lumenally
Black et al. • Translocon Proteins in Adrenal SER
exposed N-terminal domain that possesses two N-glycosylation sites (81).
In vitro translation/translocation reactions of the mRNAs were performed
in a rabbit reticulocyte lysate system (nuclease-treated lysate; Promega,
Madison, WI) supplemented with [35]S-Met at 30 C for 45 min in a total
volume of 13 ␮l. In the case of SP-CTF, the reaction was diluted with buffer
(20 mm HEPES, 100 mm NaCl) and an aliquot layered over a high salt
sucrose cushion (150 ␮l, 100 mm KCl, 2 mm MgAc2, 0.5 m sucrose). After
a brief spin (10 min at 60,000 rpm, 4 C) in a TL100 centrifuge (Beckman
Coulter, Inc., Fullerton, CA), the resulting pellet, containing microsomes
and translocated polypeptides, was resuspended in sample buffer [100 mm
Tris (pH 6.8), 4% SDS, 20% glycerol]. The remainder of the translation/
translocation reaction was precipitated with saturated ammonium sulfate
and the TCA washed pellet resuspended in sample buffer containing additional Tris to buffer any residual TCA. In the case of op-156, the entire
reaction was precipitated with saturated ammonium sulfate, washed with
TCA, and resuspended in the same sample buffer. Aliquots of each preparation were analyzed for newly synthesized polypeptides by autoradiography after SDS-PAGE.
Results
To delineate elements involved in translocation and processing
of proteins targeted for the ER, we used antibodies to the proteins
in Table 1 (listed in bold). We compared the distribution of the
immunoreactive proteins in Western blots of adrenal microsomal
subfractions with levels seen in similarly prepared microsomal
subfractions from liver and in pancreatic rough microsomes.
Microsomal subfraction protein and ribosome content
As expected, based on the morphology of the cells (Figs.
1 and 2), smooth microsomes comprised a considerably
greater percentage of the total microsomal fraction prepared
from adrenals of control animals than from the liver, whereas
Endocrinology, October 2005, 146(10):4234 – 4249
4239
FIG. 4. Ribosomal protein S3 is confined to the rough microsomes, but
high concentrations of RI, a subunit of the OST complex, are found in
smooth microsomes from the adrenal. Microsomes were separated on
a sucrose density gradient to obtain the smooth (S), intermediate
(R/S), and rough (R) microsomal fractions. Rough microsomes from
dog (D) pancreas (P) were used for comparison. Microsomal subfractions from rat (R) liver were compared with those from guinea pig (G)
liver and adrenal. Microsomal proteins from each subfraction (4 mg)
were separated on 8% SDS-polyacrylamide gels and immunoblotted
using antibody against ribosomal protein S3 and rat RI. ECL was used
to visualize the reactive proteins. The percentages of RI were calculated from the density values of the blots adjusted for the total protein
in each subfraction, using dog pancreatic rough microsomes as the
standard (100%). Values represent the mean ⫾ SEM of three microsomal preparations for guinea pig liver and four for guinea pig adrenal
and rat liver.
rough microsomes were more abundant in liver (Fig. 3)1.
However, when animals were treated with xenobiotics,
which induce the SER in hepatocytes, the liver smooth microsomal fraction increased, reaching levels comparable with
those in adrenal smooth microsomes.
Previous biochemical characterization showed that RNA
was primarily localized to the rough microsomal fraction in
these preparations, indicating that these were enriched for
bound ribosomes (65, 67, 68). To confirm the relative purity
of the microsomal subfractions used in this study with respect to bound ribosomes, antibody to the S3 ribosomal protein was used to visualize the distribution of ribosomal protein among the subfractions. Immunoreactive protein was
confined almost exclusively to the ribosome-bearing subfractions prepared from both liver and adrenal (Fig. 4). Little,
if any, immunoreactive protein was present in the smooth
microsomes, in agreement with the previous biochemical
assays.
Proteins involved in N-glycosylation, the OST
complex subunits
FIG. 3. The smooth microsomal subfraction represents over 75% of
total microsomal proteins obtained from adrenal tissue, but 25–35%
of control liver total microsomes. Treatment of animals with agents
known to increase CYPs and other enzymes involved in xenobiotic
metabolism increased the relative amount of smooth microsomes from
liver tissue 2-fold. The percent of total microsomal protein in each
microsomal fraction (S, smooth; R/S, intermediate; R, rough) was
calculated for fractions obtained from control (C) guinea pig (GP)
adrenal and for liver tissue from control (C) guinea pigs (GP) and rats.
These were compared with corresponding data derived from liver
tissue of guinea pigs treated with 3MC, known to induce CYP1A, and
rats treated with PB, known to induce CYP2B. The values shown
represent the mean ⫾ SEM of three microsomal preparations for
guinea pig liver and four for guinea pig adrenal and rat liver.
Because the ribophorins were the first proteins identified
as characteristic of RER (60), we first compared the distri1
The recovery of rough microsomes isolated by subcellular fractionation of the guinea pig adrenal is greater than what would be predicted
based on previous stereological analysis of RER and SER volumes in the
inner cortex (6). This reflects, in part, the fact that the zona glomerulosa
cells, which possess more RER than the inner cortical cells, were not
included in the morphological analysis. It also reflects an underestimate
of RER volume by the morphological analysis. Small cisternae and small
patches, or even single ribosomes, bound to tubular elements would not
have been adequately accounted for by the grid technique employed in
this stereological study.
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Black et al. • Translocon Proteins in Adrenal SER
bution of ribophorin I in liver microsomes from rat and
guinea pig with that in microsomes from guinea pig adrenal,
using dog pancreatic rough microsomes as a standard (Fig.
4). As previously reported (62), RI was a prominent component of rough microsomes from the dog pancreas, a tissue
devoted almost solely to the synthesis of secretory proteins.
Among microsomal subfractions obtained from liver of both
rat and guinea pig, RI was in highest concentration in the
rough microsomes. Its concentration was lower in the intermediate microsomal fraction, which bore fewer ribosomes,
and a small, somewhat variable amount was detectable in
liver smooth microsomes.
In contrast, in adrenal microsomal subfractions, the concentration of RI in smooth microsomes was equal to or
greater than that seen in the rough microsomes. Stripping
ribosomes from adrenal microsomes after subfractionation
increased the intensity of RI immunoreactivity, an index of
its concentration per milligram microsomal protein, in rough
microsomes by about 30%. However, the amount of this OST
subunit in the smooth microsomes remained particularly
striking. Given the fact that the smooth microsomes comprise
the bulk of the total microsomal fraction from adrenal tissue
(Fig. 3), the total amount of ribophorin I was considerably
greater in adrenal smooth than in adrenal rough microsomal
fractions (Fig. 4).
We then compared levels of other OST subunits, RII,
OST48, and DAD1, in microsomal fractions from both
tissues with those of RI (see Figs. 5 and 7). The results were
similar to those obtained for RI. In microsomal fractions
from liver, these proteins were all in highest concentration
in the rough microsomes. Yet in those from the adrenal, all
occurred in equal or greater concentration in nonribosome-bearing microsomal subfractions: smooth ⬎ intermediate ⬎ rough.
Proteins involved in ribosome targeting, binding,
translocation, and signal peptide cleavage
FIG. 5. OST complex subunits, the Sec61 core of the translocation
apparatus, and several other associated proteins, but not ribosomes
and ␣TRAP, have a concentration in guinea pig adrenal smooth microsomes equal to or greater than that in adrenal rough microsomes,
as do the molecular chaperones, BiP, Grp94, and calnexin. Microsomal subfractions were prepared as described in Materials and Methods. Proteins from dog (D) pancreatic (P) rough (R) microsomes were
compared with guinea pig (G) liver (L) rough microsomes and adrenal
rough, intermediate (R/S), and smooth (S) microsomes. After separation by SDS-PAGE, proteins were immunoblotted with antibodies
directed against OST complex subunits, RI, RII, OST48, and DAD1;
the molecular chaperones, BiP, Grp94, and calnexin; core elements of
the translocation apparatus, Sec61␣ and Sec61␤; associated proteins,
␣TRAP, ribosomal protein S3 (Ribo S3), SR␣, SPC12, Sec 62, and
Sec63; and proteins involved in sterol and steroid synthesis, HMGR,
CYP17, and 3␤HSD. With the exception of Ribo S3 and ␣TRAP, all of
the immunoreactive proteins were in equal or greater concentration
in the smooth than in the rough microsomes. For separation of the
low-molecular-weight DAD1, Sec61␤, and SPC12, 20% acrylamide
gels and tricine buffer were used. All other proteins were separated
on standard 8% acrylamide gels. In most cases, 12 ␮g of guinea pig
Sec61␣ and -␤, core components of the translocation apparatus, were in highest concentration in rough microsomes
from pancreas and liver (see Figs. 5 and 7). However, in
adrenal microsomes Sec61␣ and -␤ were detectable in similar
concentrations in the smooth and rough microsomal subfractions (Fig. 5).
SR␣ and the 12-kDa subunit of the signal peptidase complex (SPC12), representing complexes involved in targeting
of nascent chains to the ER and cleavage of signal peptides,
respectively, were more uniformly distributed among the
subfractions from both liver and adrenal (see Figs. 5 and 7).
However, ␣TRAP, one of four subunits of the TRAP complex,
which colocalizes with native ribosome-channel complexes,
was localized primarily in the ribosome-bearing subfractions
microsomal protein were loaded per lane and 6 ␮g of dog pancreatic
microsomal protein. For comparison of RI and DAD1, 4 or 24 ␮g of
protein, respectively, from each subfraction was used. In the case of
␣TRAP, microsomal proteins were loaded as follows: dog pancreatic
rough microsomes, 12 ␮g; guinea pig liver rough microsomes, 24 ␮g;
and all subfractions from guinea pig adrenal, 48 ␮g. Reactive proteins
were visualized by ECL in most cases. Proteins reacting with anti-RII,
OST48, SR, CYP17, and 3␤HSD were visualized using DAB.
Black et al. • Translocon Proteins in Adrenal SER
Endocrinology, October 2005, 146(10):4234 – 4249
4241
from both adrenal and liver (see Figs. 5 and 7). Little ␣TRAP
was detected in the smooth microsomes obtained from either
organ.
Sec62 and Sec63, mammalian homologs of proteins essential for posttranslational translocation in yeast, were present
in both the rough and smooth microsomal subfractions from
liver and adrenal (data not shown).
Molecular chaperones
We examined several proteins involved in folding of
newly synthesized polypeptides and ER quality control, generally considered to be distributed throughout the ER: two
lumenal proteins, BiP and GRP94, and one membrane protein, calnexin (see Table 1). In liver microsomes, particularly
those of the guinea pig, BiP and GRP94 were in higher concentration in the rough microsomes (see Fig. 7). In adrenal
microsomal subfractions, their concentrations in smooth microsomes were equal to or slightly greater than in rough
microsomes (Fig. 5). Calnexin was present in all microsomal
subfractions from liver but did not show a consistent differential pattern of distribution (see Fig. 7). In adrenal microsomes, the levels of calnexin were higher in the smooth
microsomes (Fig. 5).
Enzymes involved in sterol and steroid synthesis
Having found high levels in adrenal smooth microsomes
of all translocon subunits and associated elements examined,
except for ribosomal protein and ␣TRAP, it seemed important to confirm that preferential localization in smooth microsomes of enzymes involved in sterol and steroid synthesis
was retained in these subcellular fractions. Therefore, we
examined the levels of HMGR and two enzymes involved in
steroid synthesis, 3␤HSD and 17␣-hydroxylase, a cytochrome P450 (CYP17). All of these enzymes were in highest
concentration in the adrenal smooth microsomes (Fig. 5).
None were detectable in corun rough microsomes from liver
or pancreas, although HMGR was present in liver smooth
and intermediate microsomes (data not shown).
Immunocytochemistry
To determine whether the translocon components and
steroidogenic enzymes have a similar distribution in situ,
immunocytochemistry was performed on isolated adrenocortical cells. These cells retain their abundant SER in vitro,
particularly in the presence of ACTH (74). Both CYP17 and
Sec61were distributed fairly evenly throughout the tubular
ER network of the isolated cells (Fig. 6, A and B, respectively).
Corun controls showed minimal staining (Fig. 6C). Little
change in the distribution of CYP17 and Sec61 was detected
in ACTH-treated cells at this resolution (data not shown).
However, in some cells, hotspots of intense labeling occurred
for both proteins, which seemed to be more abundant in
ACTH-treated cells (data not shown). More detailed analysis
FIG. 6. Immunocytochemistry revealed distribution of both CYP17
and Sec61 throughout the predominantly smooth-surfaced tubular
ER network. Adrenocortical cells were isolated and maintained in
vitro and immunocytochemistry performed, as described in Materials
and Methods. Antibody localization was visualized by confocal microscopy. A, Distribution of CYP17 throughout the tubular ER. Bar,
7.5 ␮m ⫻ 1300. B, A similar distribution for Sec61. Bar, 5 ␮m ⫻ 2000.
C, Minimal staining in corun controls incubated with the fluoresceinconjugated secondary antibody alone. Bar, 20 ␮m ⫻ 360.
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Black et al. • Translocon Proteins in Adrenal SER
will be required to see whether these correspond to the
organized arrays of SER observed in these cells with the
electron microscope (4 – 6).
Liver microsomal subfractions from animals treated
with xenobiotics
In an attempt to see whether the distribution of OST subunits,
translocon components, and chaperones in adrenal microsomes
was simply related to the larger amount of SER in adrenocortical cells, we prepared liver microsomal subfractions from animals treated with PB and 3MC. These compounds are known
to induce the cytochrome P450s (CYPs) that metabolize them,
CYPs 2B and 1A, respectively, as well as other enzymes involved in metabolism of foreign compounds. These enzymes
are located predominantly in the SER, and their induction leads
to an increase in the amount of SER in hepatocytes (for review
see Ref. 8). In the treated animals, the relative amount of protein
in the smooth microsomal fraction reached levels comparable
with adrenal smooth microsomes (Fig. 3). Each xenobiotic agent
induced the appropriate CYP: PB treatment resulted in increased levels of CYP2B and 3MC treatment resulted in increased levels of CYP1A, particularly in the smooth microsomes
(Fig. 7).2
The increase in SER was relatively specific for enzymes
involved in xenobiotic metabolism. There was a slight increase in the concentration of HMGR in all liver microsomal
subfractions from treated animals and the concentration of
the molecular chaperones, BiP and GRP94, did shift toward
the smooth microsomal fraction in treated animals (Fig. 7).
However, although there was some shift of the OST complex
subunits, as well as Sec61␣ and -␤ proteins toward the
smooth microsomal fraction, their increases in the lighter
fractions were variable, and none achieved the high levels
seen in adrenal microsomes (Fig. 7).
Adrenal microsomal subfractions from other species
To eliminate the possibility that the presence of transloconassociated proteins in adrenal smooth microsomes was confined to the guinea pig, we examined similarly prepared
microsomes from adrenals of several species: rat, dog, cattle,
rabbit, sheep, and pig. In most, as in the guinea pig adrenal,
OST and Sec61 complex subunits as well as the molecular
chaperones BiP, GRP94, and calnexin, were found to be in
equal or greater concentration in smooth microsomes compared with rough microsomes (Fig. 8). The sole exceptions to
the equal or greater concentration of these proteins in the
smooth microsomes were in the rat adrenal. In the rat adrenal, the OST components as well as BiP and GRP94 were
in greater concentration in the rough microsomes. In all of the
species examined, however, the SER markers CYP17 and
3␤HSD were in greater concentration in the smooth microsomes than in rough microsomes. In all cases, the ribosomal
2
The proliferation of SER coincident with induction of CYP1A observed here in guinea pig liver differs from previous reports for rodent
liver and 293T cells (see Ref. 8). In those experiments, increases in CYP1A
were not accompanied by increases in the SER. This suggests that there
may be differences in the effects of aromatic hydrocarbons on CYP1A
induction and consequent SER proliferation, depending on the species
or cell type and specific substance used.
FIG. 7. Treatment with xenobiotic agents known to cause proliferation of the SER in hepatocytes did not produce a proportional shift of
OST and Sec61 components into liver smooth microsomal subfractions, although a shift of BiP and Grp94 toward this subfraction was
observed. Rats were treated with PB, which induces CYP2B, and
guinea pigs with 3MC, which induces CYP1A. Microsomal subfractions were prepared from liver tissue as described in Materials and
Methods. Proteins (12 ␮g) from rough (R), intermediate (R/S), and
smooth (S) microsomes of control (⫺) and treated (⫹) animals were
separated by SDS-PAGE and immunoblotted with antibodies made
against OST components, molecular chaperones, and elements of the
translocation apparatus, as in Fig. 5, and antibodies against the
cytochrome P450s CYP2B, induced by PB, and CYP1A, induced by
3MC. For separation of the lower-molecular-weight proteins, DAD1,
Sec61␤, and SPC12, 20% acrylamide gels and tricine buffer were used.
For separation of other proteins, standard 8% gels were used. In most
cases, ECL was used to detect reactive proteins. DAB was used for
detection of RI, RII, BiP (rat), and ␣TRAP (rat).
protein S3 was localized to the ribosome bearing fractions
(data not shown). This gives increased confidence that the
broader distribution of translocon-associated proteins in the
ER is a property of most adrenocortical cells and perhaps of
steroid-secreting cells in general.
Oligosaccharyltransferase activity
To investigate whether the OST subunits in adrenal
smooth microsomes form a complex active in N-glycosyla-
Black et al. • Translocon Proteins in Adrenal SER
FIG. 8. The distribution of OST complex subunits, Sec61 and molecular chaperones among microsomal subfractions from adrenal of several other species, is similar to that seen in guinea pig adrenal microsomes: smooth ⬎ rough. Microsomal subfractions were prepared as
described in Materials and Methods. Rough (R), intermediate (R/S),
and smooth (S) microsomes from sheep (not shown), pig, rabbit, bovine, dog, and rat adrenals were compared. Microsomal proteins were
separated by SDS-PAGE and immunoblotted with antibodies made
against OST components, molecular chaperones, elements of the
translocation apparatus, and steroidogenic enzymes as in Fig. 5. In
most cases, 12 ␮g of microsomal protein were loaded in each lane and
separated on 8% gels. For DAD1, 18 ␮g of microsomal protein from
each subfraction was used, and both DAD1 and Sec61␤ were separated on 20% acrylamide gels, using tricine buffer. Detection of reactive proteins was by DAB for RI and ECL for RII, OST48, and DAD1.
tion we assayed for oligosaccharyltransferase activity using
an OTP containing the N-glycosylation acceptor sequence,
Asn-Tyr-Thr (76). In this assay, adrenal smooth microsomes
had a capacity for N-glycosylation at least equivalent to that
of dog pancreatic microsomes (Fig. 9A). It was greater than
the capacity for N-glycosylation by the adrenal intermediate
smooth/rough or rough microsomes. Omission of activated
sugars did not significantly change these results, indicating
that there are dolichol-linked oligosaccharides already
present in the adrenal microsomes (data not shown). Given
the levels of OST components detectable by immunoblotting
in the adrenal rough microsomes, the activity measured in
these microsomes was disproportionately low. This result
was very reproducible and may accurately reflect the composition of these microsomes; e.g. some lack of other element(s), as yet undefined, necessary for full activity. However, we cannot exclude the possibility that the low activity
resulted from some artifact of preparation.
To investigate whether the glycosylated products formed
were similar in adrenal and pancreatic microsomes, we re-
Endocrinology, October 2005, 146(10):4234 – 4249
4243
FIG. 9. Adrenal smooth microsomes have high oligosaccharyltransferase activity. A, The activity for N-glycosylation in adrenal smooth
microsomes was comparable with that in dog pancreatic microsomes.
Data for glycosylation of the OTP acceptor peptide are shown at the
top of the figure as the mean ⫾ SE of at least five samples. B, Separation of OST assay products by TLC confirmed that both pancreatic
and adrenal microsomes formed slower migrating products typical of
the glycosylated OTP. However, there were differences in the migration of the products produced. Dog pancreatic microsomes (DP) favored formation of slightly faster migrating products, whereas guinea
pig adrenal microsomes (S, smooth; R/S, intermediate; R, rough) favored formation of more slowly migrating products. The nonglycosylated OTP substrate is shown for comparison. C, Enzymatic digestion
indicated that the differences in migration on TLC reflected structural differences in the products. After incubation with Endo H (E),
the products from both pancreas and adrenal were completely digested to the OTP-GlcNAc forms, confirming that N-glycosylation had
occurred. However, when digested with ␣-mannosidase (M), pancreatic microsomal products were almost completely digested to OTPGlcNac2␤Man, whereas adrenal products were largely resistant to
␣-mannosidase digestion, suggesting that glucosidase(s) were not as
active in adrenal microsomes.
solved the products of N-glycosylation by TLC. When eluates of the microsomal incubations were subjected to TLC,
the radiolabeled material migrated with the low Rf typical of
the glycosylated OTP (76, 77) (Fig. 9B). There were, however,
some differences in the TLC patterns of products formed by
pancreatic and adrenal microsomes. Of the two major spots
resolved, the upper one was preferentially visible in dog
pancreatic microsomes, whereas the lower was more prom-
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Endocrinology, October 2005, 146(10):4234 – 4249
inent in the adrenal smooth microsomes [Fig. 9, B (lanes DP
and S) and C (pancreas and adrenal, lane C)].
We then examined the nature of the glycosyl chain more
closely by enzymatic digestion. Endoglycosidase H (EndoH)
cleaves all N-linked carbohydrate chains normally occurring in
the ER between the two innermost GlcNAc residues, leading to
very fast migration of the digested product. EndoH digestion
was used to confirm that the isolated glycotripeptides contain
typical N-linked carbohydrate chains (Fig. 9C, pancreas and
adrenal, lane E). In contrast, Jack bean ␣-mannosidase is an
exomannosidase that cleaves only terminal ␣-linked mannoses.
In dog pancreas-derived glycotripeptides, incubation with this
enzyme led to a complete digestion down to the GlcNAc2␤Man
core (Fig. 9C, pancreas, lane M). In contrast, when glycotripeptides derived from adrenal smooth microsomes were subjected to this digest (Fig. 9C, adrenal, lane M), an additional spot
with lower Rf value was seen. This partially resistant material
presumably retains one or more of the terminal glucose residues that are initially present on the transferred N-glycosyl
chain (Glc3Man9GlcNAc2). This assumption was confirmed by
comparison with glycotripeptides generated in the presence of
castanospermine, a glucosidase inhibitor (data not shown).
Thus, the slower migration of adrenal-derived material can be
explained by a low activity of at least one glucosidase (I and/or
II) in adrenal microsomes. If glucosidase II were preferentially
affected, retention of monoglucosyl residues could result in
more extended association with chaperones such as calnexin,
abundant in the smooth microsomes, facilitating retention of
glycosylated proteins in the SER (56, 57).
Ribosome targeting and binding
Having found that the OST components formed a functional
unit in adrenal smooth microsomes, we then sought to determine whether the SR and Sec61 complexes function in this
setting. To determine SR activity, we took advantage of a previously developed photocross-linking assay in which one can
assess SR activity by looking for a decrease in the amount of
signal peptide-associated SRP54 (23). Truncated RNCs for
86pPL containing photocross-linker-modified lysines, were
prepared in a reticulocyte lysate system. In absence of microsomal membranes a 62-kDa photoadduct formed representing
the 86pPL signal peptide cross-linked to SRP 54 kDa (24, 25).
Addition of SR-containing dog rough microsomes reduces the
intensity of the SRP cross-link due to the release of SRP from the
nascent chain by SR present in the microsomal membranes (23).
In this assay, adrenal smooth microsomes were capable of decreasing the intensity of the SRP cross-link, although not to the
same extent as dog pancreatic microsomes (Fig. 10A). Because
the levels of SR␣ are lower in adrenal smooth microsomes than
in liver or pancreas rough microsomes, it is possible that some
ribosome-nascent chain-SRP complexes interacted with SR-deficient Sec61 complexes and were therefore unable to release
SRP.
To assay ribosome binding, high salt-stripped 86pPL
RNCs were incubated with either dog pancreatic rough microsomes or adrenal smooth microsomes and membrane
bound vs. unbound ribosomes subsequently separated on a
sucrose gradient (78). Membranes with bound RNCs float to
Black et al. • Translocon Proteins in Adrenal SER
FIG. 10. Adrenal smooth microsomes have functional SRs and ribosome binding sites. A, Dog pancreatic and adrenal smooth microsomes
harbor functional SRs. Radiolabeled ([35]S-Met) RNCs for a truncated
form of preprolactin (86pPL) containing photocross-linker-modified
lysines were produced by in vitro translation as described in Materials
and Methods. After incubation of the RNCs in the presence and
absence of puromycin/KOAc washed dog pancreas microsomes (p
PKRM) or adrenal smooth microsomes (ad SM), samples were irradiated (h x v) to induce cross-linking. In the absence of microsomes,
a band representing the cross-linked 86pPL and SRP 54-kDa subunit
was present in the irradiated samples (⫹) but not in samples that had
not been irradiated (⫺). In the presence of both sets of microsomes,
the intensity of the 86pPL ⫻ SRP54 band was diminished, indicating
that membrane-associated SR had mediated the release of SRP from
the signal peptide. B, Adrenal smooth microsomes have functional
ribosome binding sites. [35]S 86pPL high salt-washed RNCs were
prepared as described in Materials and Methods and 5 ␮l aliquots
incubated in the presence or absence of the indicated volumes (microliters) of puromycin/KOAc washed dog pancreas microsomes (p
PKRM) (10 mg/ml) or adrenal smooth microsomes (ad SM) (12 mg/ml).
Bound RNCs were separated by flotation (flot.) of the membranes with
associated RNCs in a discontinuous sucrose gradient. The top fractions were collected and analyzed for RNC content by autoradiography after SDS-PAGE (lanes 3–5). In parallel, top fractions (lanes 3–5)
or the indicated amounts of membranes (lanes 1–2) were analyzed by
Western blotting for Sec61␣ and OST48. Both sets of microsomes have
functional ribosome binding sites. The amount of RNCs bound to
adrenal smooth microsomes increased roughly in proportion to the
amount of microsomal protein included in the assay and to the Sec61␣
and OST content of the membranes (lanes 4 –5).
the top of the gradient, whereas the bottom fraction contains
free, untargeted ribosomes. In this assay, adrenal smooth
microsomes bound RNCs as did stripped dog pancreatic
rough microsomes. To normalize the data, we compared the
binding to the levels of Sec61 and OST48 in adrenal smooth
microsomes at two levels of microsomal protein. The binding
increased in proportion to the increase in microsomal protein
and the levels of OST48 and Sec61 (Fig. 10B).
Black et al. • Translocon Proteins in Adrenal SER
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4245
Cotranslational translocation, signal peptide cleavage, and
N-glycosylation
To confirm that the activities of translocon, SPC and OST
components could be coordinated in adrenal smooth microsomes, we assayed the ability of adrenal microsomal subfractions to translocate peptides synthesized on ribosomes
bound to these membranes as well as to cleave signal peptides and N-glycosylate incoming, newly synthesized
polypeptides. The adrenal smooth microsomal subfractions
were at least as capable of these functions as adrenal rough
microsomal subfractions (Fig 11, A and B, respectively).
However, the levels of both activities were considerably
lower than in dog pancreatic rough microsomes. Comparison of the levels of N-glycosylation of the opsin fragment
with the levels of oligosaccharyltransferase activity measured using the OTP acceptor (Fig. 9A), suggest that the
amount of processing observed reflects the rate of targeting
and translocation of the newly synthesized heterologous
polypeptides rather than the full capacity of these enzyme
complexes.
Discussion
The remarkable finding presented in this paper is the
discrepancy between the morphological appearance and the
biochemical properties of the ER in adrenocortical cells with
regard to elements involved in protein synthesis, translocation, and processing. High levels of Sec 61␣ and -␤, key
components of the translocation channel, as well as subunits
of the OST complex, which is involved in N-glycosylation of
newly synthesized polypeptides, are found in adrenal
smooth microsomes, derived largely from the abundant SER
in these cells. These proteins have been considered to be
localized primarily in ribosome-bearing rough microsomes
and to be markers of the RER (60 – 63), yet in adrenocortical
cells they are distributed throughout the tubular ER network,
which is predominantly smooth surfaced. This discrepancy
forced reevaluation of the nature of the SER and its distinction from RER in steroid-secreting cells as well as the functions of OST and Sec61 in membranes thought to be devoid
of de novo protein synthesis.
Despite the broader distribution of Sec61 and OST complex proteins in adrenal microsomes, two features distinguished isolated adrenal rough from smooth microsomes:
bound ribosomes and ␣TRAP. These features are shared with
rough microsomes prepared from pancreas and liver and are
consistent with active cotranslational translocation (30).
However, it seems that there are at least two forms of SER.
One, found in steroid-secreting cells, possesses abundant
machinery for the translocation and processing of proteins
targeted for the ER. The other, characteristic of protein-secreting cells, does not. The high levels of these proteins in
adrenal smooth microsomes cannot be attributed to contamination by rough microsomes. The small amount of RER
present in adrenocortical cells and the clear localization of
ribosomal protein to the rough microsomal fraction preclude
this as does their distribution throughout the ER visualized
by immunocytochemistry. Furthermore, the broader distribution of these proteins in adrenal ER is not related to the
amount of SER per se. Smooth microsomes from livers of
FIG. 11. Adrenal smooth microsomes are capable of cotranslational
translocation, signal peptide cleavage, and N-glycosylation of newly
synthesized polypeptides. A, mRNA encoding the 99-amino-acid CTF
of amyloid precursor protein linked to the bovine pPL signal peptide
sequence was translated in a rabbit reticulocyte lysate system supplemented with [35]S-Met, in the presence and absence of dog pancreatic rough microsomes (DP; 1 ␮l) and guinea pig adrenal microsomal subfractions (rough, R; intermediate, R/S; and smooth, S) (3 ␮l).
The membranes were isolated through a 0.5 M sucrose cushion to
separate unbound RNC complexes from those bound to microsomal
membranes. Microsomal proteins were separated on 12% gels and
visualized by autoradiography. The band present in reactions containing microsomes but not in controls containing only the mRNA
(arrow) represents the C-terminal polypeptide after cleavage of the
bovine pPL signal peptide. Because cleavage of the signal peptide
occurs after translocation, these data show that cotranslational translocation and signal peptide cleavage occur in adrenal smooth microsomes. B, Truncated mRNA for bovine opsin was translated in a
rabbit reticulocyte lysate system in the presence and absence of dog
pancreatic (DP) rough microsomes and guinea pig adrenal microsomal subfractions (R, rough; R/S intermediate; and S, smooth) (1 ␮l).
Aliquots of ammonium sulfate precipitates of the reactions were separated on 15% gels and the proteins visualized by autoradiography of
incorporated 35S-Met. The band present in reactions containing microsomes, but not in control reactions containing only the mRNA,
represents the N-glycosylated product (g-op-156) of the translocated
opsin fragment (op-156). The glycosylated nature of the product was
confirmed by EndoH digestion (data not shown). Based on the lumenal location of OST, we conclude that glycosylation indicates translocation of the amino terminal domain.
animals treated with xenobiotics that expand the SER in
hepatocytes did not contain high levels of these proteins.
Although not as dramatic as the data presented here, there
have been a few previous reports of smooth endomembrane
systems possessing RER-specific proteins in other cell types.
Small amounts of translocon-associated proteins have been
seen in SER found in mammalian dendrites (Sec61␣) (82) and
Caenorhabditis elegans neurites [ribosome-associated membrane protein 4 (RAMP4), TRAP␤, translocating-chain associating membrane (TRAM)] (83). However, as shown in C.
elegans, the bulk of the ribosomes and translocon-associated
proteins colocalize in the neuron cell bodies that contain
abundant RER (83). In studies using less specific reagents, an
antibody made against RER membrane proteins reacted with
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Endocrinology, October 2005, 146(10):4234 – 4249
the sarcoplasmic reticulum in skeletal muscle and smoothsurfaced cisternal stacks in Purkinje neurons (84, 85). These
compartments both contain high levels of specific multispanning transmembrane (TM) proteins (Ca2⫹ATPase and inositol 1,4,5-trisphosphate receptor, respectively) (for discussion of these and other ER domains, see Ref. 86).
We hypothesize that the components of the translocation
apparatus found in adrenal smooth microsomes participate
in synthesis of membrane proteins targeted for particular
domains in the ER and/or quality control of these proteins.
Morphologically distinct domains clearly exist in adrenocortical cells (6, 7) and smaller domains of enzyme complexes
have been postulated (87). Two lines of evidence suggest
some correlation with cholesterol production. First, translocation components were more prominent in adrenal smooth
microsomes of species in which the adrenals synthesize a
large proportion of their own cholesterol (88 –91). They were
less prominent in SER of the rat adrenal that depends on
plasma lipoproteins, rather than de novo synthesis, for cholesterol used in steroid synthesis (89, 90). Second, Sec61 and
OST were not as prominent in the equally abundant SER
induced in hepatocytes by xenobiotics. These SER membranes showed relative increases in CYPs involved in metabolism of foreign compounds but did not show a relative
increase in the concentration of HMGR.
We have demonstrated that Sec61, OST, and other components of the translocation apparatus in the adrenal SER
form functional complexes capable of ribosome binding,
translocation, signal peptide cleavage, and N-glycosylation.
These data confirm and extend an earlier report from Boime
and colleagues (92) and our own preliminary report (93).
Taken together, they strongly suggest that the adrenal SER
is potentially capable of ER-targeted protein synthesis.
Steroid-secreting cells are rich in membrane proteins involved in sterol and steroid synthesis that are potential substrates for such activity, e.g. cytochromes P450 and HMGR as
well as sterol regulatory element-binding protein (SREBP),
SREBP cleavage-activating protein (SCAP), and Insig-1, proteins involved in the sterol sensitive regulation of the levels
of HMGR and other enzymes involved in the cholesterol
biosynthetic pathway (94 –97). When adrenals are stimulated
with ACTH, HMGR activity and protein content in the SER
increase dramatically (11). The increase in HMGR activity
parallels that of cytochromes P45017␣ and -21, proteins involved in steroidogenesis. Coincident with these increases in
enzyme activity, the volume of the SER, as assessed by stereological analysis, increases 2-fold, occupying up to 70% of
the cytoplasmic volume (6). The SER also undergoes dramatic changes in morphology, often forming large complex
arrays (6).
The RER does not undergo such dramatic changes. RER
volume increases but remains sparse, occupying less than
0.8% of the cytoplasmic volume, and no significant changes
occur in its morphology (6). It remains predominantly as
ribosomes bound on short isolated cisternae or in patches
scattered along predominantly smooth-surfaced tubules.
Longer RER cisternae, although more easily identified, are
relatively rare in control and ACTH-treated cells. These morphological data are consistent with data extracted from our
previous biochemical studies (65, 66, 98). Although there is
Black et al. • Translocon Proteins in Adrenal SER
an increase in the percentage of the ribosome-bearing subfractions in the total microsomal fraction with ACTH treatment (from 32 to 40%), the majority of the increase is in the
intermediate subfraction (from 23 to 30.5%), which would be
derived from the dispersed elements, less densely covered
with ribosomes.
One model that reconciles the relatively sparse, widely
distributed RER elements seen in adrenocortical cells and
their nonconcordant increase in comparison with the SER
with the distribution of proteins involved in translocation
and processing throughout the ER is that protein synthesis
on membrane-bound ribosomes can potentially occur at multiple points on the tubular ER membranes of these cells but
occurs only in a small fraction at any one time. The rough
microsomal fraction then represents a snapshot of where
cotranslational translocation was occurring at the time of
fractionation. Although some rough microsomes could be
derived from RER devoted to protein synthesis, the majority
would be from microsomes that are rough protemp. The
Sec61 and associated complexes present in the smooth microsomes would represent translocon components awaiting
arrival of targeted ribosome/nascent chain complexes. Stimulation of synthesis would increase the number of protemp
ribosome bearing complexes, but their sum over the period
of induced membrane synthesis would be greater that that
captured at any one moment. Viewed from this perspective,
the elements involved in translocation and processing found
throughout the SER in steroid-secreting cells would be functionally similar to those seen in the RER of protein-secreting
cells but dynamically distinct.
However, the broad distribution of translocation apparatus and oligosaccaryltransferase proteins in the SER may
suggest that they have additional functions in this site. The
high levels of BiP, GRP94, and calnexin in the SER are
consistent with the functioning of this compartment not
only in protein folding and assembly but also in regulated
forms of endoplasmic reticulum-associated degradation
(50 –52). The highly regulated degradative pathways of
HMGR and the degradation of some cytochrome P450s
occur via the ubiquitin proteasome pathway (99 –101). Because proteosomes are in the cytosol, this requires retrograde transport of the protein out of the ER. The Sec61
channel complex has been implicated in retrograde translocation (14, 15). Recent studies show that the membrane
protein Derlin-1 is required for retrograde translocation of
membrane proteins (102, 103). It is linked via another
recently discovered protein VIMP [VCP (valosin-containing
protein)/p97-interacting membrane protein] to the cytosolic
AAA ATPase, p97, which is essential for movement of polypeptides from the membrane to the cytosol (102). The involvement
of Sec61 vs. Derlin-1 may depend on the endoplasmic reticulum-associated degradation substrate (104). More speculative is
a role for OST components in quality control. Recently, RI and
other OST complex proteins have been found tightly associated
with specific membrane proteins after their integration into the
membrane (105). In one case, US11, the protein also associates
with p97 and the ER chaperones BiP and calnexin (106), suggesting a link between the OST complex and quality control
processes (105). Yeast OST subunits, OST3 and OST6, and their
mammalian homologs, N33 and implantation-associated pro-
Black et al. • Translocon Proteins in Adrenal SER
tein, contain thiroedoxin domains. It has been suggested that
they may be involved in disulfide oxidoreductase regulatory
mechanisms (105). This could be important if retention and/or
release of ER proteins is dependent on disulfide bonds with
associated proteins.
In conclusion, the abundant SER in steroid-secreting cells
contains high levels of proteins involved in translocation and
processing of ER-targeted proteins. In these cells, ribosomes are
scattered in patches along the predominantly smooth-surfaced
tubular network. This form of RER increases when the cells are
treated with ACTH. We suggest that these smaller RER elements represent transitory complexes between ribosomes and
the translocation apparatus participating in local synthesis of
TM proteins involved in sterol and steroid synthesis within
specific domains of the ER (see Refs. 13, 107, and 108 for review
of TM protein synthesis) as well as proteins involved in membrane protein folding and/or identification of misfolded proteins and their retrograde transport (104). Targeting of the translocation substrates to specific ER domains could involve not
only SRP and SR but also mRNA trafficking (109) and signal
sequence information (110, 111) as well as cytoplasmic factors
and the membrane protein and lipid composition of the ER
domains (111). The equilibrium of bound ribosomes in any one
domain could be determined not only by the rate of synthesis
and competition for synthesis of cytoplasmic proteins, as suggested by Potter and Nicchitta (112), but also by competition for
retrograde transport of proteins out of the ER. Alternative roles
in synthesis vs. degradation could be regulated by levels of
cholesterol (113) or other lipid moieties (114) in the ER membranes, determined, in part, by levels of trophic hormones.
Acknowledgments
The authors gratefully acknowledge assistance of Tellervo Huima in
electron microscopic analysis; Heide Plesken, Jody Culkin, and Robert
Boyd in graphics and photography; and advice from Carmen DeLemos
Chiarindini in immunofluorescence and Andrei Nokonov in confocal
microscopy. They also express their appreciation to all the individuals
who provided antibodies used in these analyses; Jean Barilla, whose
early identification of the ribophorins in adrenal smooth microsomes on
the basis of size has now been confirmed; and Dr. Y. Yu, who prepared
the stripped microsomal membranes.
Received March 30, 2005. Accepted May 27, 2005.
Address all correspondence and requests for reprints to: Virginia H.
Black, Department of Cell Biology, New York University School of
Medicine, 550 First Avenue, New York, New York 10016. E-mail:
[email protected].
This work was supported in part by National Institutes of Health
Grants HD04005, AG01468, AM32862, DK39671, HL48476, ES00260, and
ACS BC-593 (to V.H.B.) and American Cancer Society RPG-92-009-CB (to
G.K.).
Present address for A.S.: Department of Anatomy and Cell Biology,
Temple University, Philadelphia, Pennsylvania.
Present address for K.v.L.: Department of Radiology, Neuroprotection Research Laboratory, Massachusetts General Hospital, Charlestown, Massachusetts.
Results from this work were presented in part at the Annual Meeting
of the Society for Cell Biology, San Francisco, CA, December 2000 and
2002, and the IX Conference on the Adrenal Cortex, San Francisco, CA,
June 2002 (93).
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