Download Identification and characterization of AtCASP, a plant

 Springer 2005
Plant Molecular Biology (2005) 58:109–122
DOI 10.1007/s11103-005-4618-4
Identification and characterization of AtCASP, a plant
transmembrane Golgi matrix protein
Luciana Renna1, , Sally L. Hanton1, , Giovanni Stefano1, , Lauren Bortolotti1, Vikram
Misra2 and Federica Brandizzi1,*
1
Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada;
(*author for correspondence; e-mail [email protected]); 2Department of Veterinary Sciences, 52
Campus Drive, University of Saskatchewan, Saskatoon, SK S7N 545B, Canada; These authors have
contributed equally to the work
Received 14 February 2005; accepted in revised form 28 March 2005
Key words: CASP, membrane Golgi matrix proteins, plants
Abstract
Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary
for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we
report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long
N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with
amino acid residues which are highly conserved across species. Through fluorescent protein tagging
experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of
this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition,
we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However,
mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER
export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its
correct localization in plant cells. These data suggest that mammalian and plant cells have different
mechanisms for concentrating CASP in the Golgi apparatus.
Introduction
The Golgi apparatus is a vital organelle dedicated
to processing and sorting of secretory cargo in all
eukaryotic cells (Palade, 1975). In plants, vesicles
containing protein cargo are targeted to both the
cis- and trans-cisternae from other organelles
(reviewed by Jürgens, 2004). It is likely that vesicles
also move between cisternae as may occur in other
species (Volchuk et al., 2004). How the specificity
of vesicular traffic between individual cisternae is
conferred has not yet been well-characterized in
plants, although a considerable amount of evidence
regarding the function and localization of SNAREs
(Sanderfoot et al., 2001; Uemura et al., 2004) and
Rabs (Drew et al., 1993; Ueda et al., 1996; Batoko
et al., 2000; Inaba et al., 2002; Saint-Jore et al.,
2002; Grebe et al., 2003) in the Golgi apparatus has
been presented. Further investigations based on
those already carried out will doubtless clarify the
molecular mechanisms involved.
Plant cells have numerous Golgi stacks dispersed in the cytoplasm (Boevink et al., 1998;
Nebenführ et al. 1999). Each stack contains cis-totrans cisternae and moves on an underlying
membrane network, the endoplasmic reticulum
(ER), by means of actin-myosin motors (Boevink
et al., 1998; Brandizzi et al., 2002a; Saint-Jore
110
et al., 2002). The Golgi apparatus and ER export
sites (ERES, daSilva et al., 2004) form secretory
units that move together, although the ER to
Golgi transport of cargo molecules is independent
of the movement (Brandizzi et al., 2002a). It is
currently unknown how the plant Golgi apparatus
maintains its structure despite the rapid transport
of proteins to and from this organelle (Brandizzi
et al., 2002a) and while it is moving over the ER
(daSilva et al., 2004).
It has been proposed that the mammalian
Golgi apparatus may maintain its form and ensure
vesicular trafficking via a proteinaceous structure
known as the matrix or scaffold (Slusarewicz et al.,
1994). Matrix proteins are involved in binding to
and linking adjacent membranes in vesicle docking
during protein transport, and linking cisternae
during Golgi stack formation (Sonnichsen et al.,
1998; Shorter and Warren, 1999).
Long coiled-coil proteins such as golgins are
components of this matrix. Golgins were initially
identified on the Golgi and on endosomes, mostly as
auto-antigens in autoimmune disorders (Dohlman
et al., 1993). Based on their restricted intracellular
distributions and their predicted rod-like structure,
it has been proposed that these proteins play a role
in tethering vesicles to target Golgi membranes
prior to fusion. However, such proteins may also
play a structural role as scaffolds for the assembly of
other factors important for fusion, such as Rab
proteins (reviewed in Barr and Short, 2003).
The large number of different golgins suggests
that they may perform more than one general
function in the Golgi. Indeed, tethering and
organizational functions for a number of golgins
have been shown in in vitro systems. For instance,
Uso1p and its mammalian homologue p115 are
required for ER-to-Golgi transport in vivo, and
reconstitution of this process in vitro has shown
that the requirement is at the stage of vesicle
tethering to the cis-Golgi (Cao et al., 1998;
Seemann et al., 2000; Alvarez et al., 2001). Two
other coiled-coil proteins, GM130 and giantin,
have been shown to recruit p115 to Golgi membranes (Sonnichsen et al., 1998; Lesa et al., 2000;
Puthenveedu and Linstedt, 2001). These proteins
also seem to contribute to cisternal structure
in vivo and are required in an in vitro assay system
that reconstitutes the stacking of Golgi cisternae
(Shorter and Warren, 1999; Puthenveedu and
Linstedt, 2001).
Most golgins are peripheral membrane proteins
and often have short non-coiled-coil regions at
either end of the protein that mediate targeting
and other interactions. For example, the C-terminus
of GM130 binds to GRASP65, a lipid-anchored
protein on the cis-Golgi (Barr et al., 1998).
Similarly, several golgins share a C-terminal GRIP
domain that is sufficient to target them to the
trans-Golgi (Barr, 1999; Kjer-Nielsen et al., 1999;
Munro and Nichols, 1999; Barr and Short, 2003).
In contrast, three golgins, CASP (CCAAT-displacement protein alternatively spliced product),
giantin and golgin-84 have been experimentally
proved to be integral membrane proteins anchored
to the bilayer via a C-terminal transmembrane
domain (Linstedt and Hauri, 1993; Bascom et al.,
1999; Misumi et al., 2001; Gillingham et al., 2002).
Ultrastructural evidence points towards the
existence of a Golgi matrix in plant cells (Staehelin
and Moore, 1995; Neumann et al., 2003) and the
existence of DNA sequences in the Arabidopsis
genome encoding homologues of mammalian
matrix proteins has been reported (Gillingham
et al., 2002; Rose et al., 2004). However, to date
there is little data on the localization and dynamics
of these proteins. Only recently, the localization of
a green fluorescent protein fusion to the GRIP
domain of an Arabidopsis gene (AtGRIP) has been
described (Gilson et al., 2004). The C-terminal
domain from AtGRIP is sufficient to target the
Golgi apparatus in plant and mammalian cells,
indicating that GRIP-proteins might be implicated
in a targeting mechanism that is conserved amongst
eukaryotes (Gilson et al., 2004). However, apart
from the identification of genomic sequences, no
other data are available on integral membrane
Golgi matrix proteins in plants.
Here we have studied a coiled-coil integral
membrane protein from Arabidopsis thaliana
(AtCASP) that shares a high degree of homology
with human and yeast CASP, a known golgin in
these organisms (Gillingham et al., 2002; Mansour
et al., 2002; Barr and Short, 2003). AtCASP shows
an overall domain structure that is strongly
conserved across kingdoms. By means of fluorescent protein tagging, we have determined that
AtCASP targets the Golgi apparatus in plant cells
and also contains encoded information to target
the Golgi apparatus in mammalian cells. Our
results indicate that integral membrane Golgi
matrix proteins exist in plant cells and belong to
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a highly conserved family that shares overall
structure and targeting domains with homologues
in other kingdoms.
Materials and methods
Molecular Cloning
Standard molecular techniques were used as
described in Sambrook et al. (1989). The fluorescent proteins used in this study were based on
fusions with either mGFP5 (Haseloff et al., 1997)
or EYFP (Clontech Inc., California, USA). The
spectral properties of mGFP5 allow efficient spectral separation from YFP (Brandizzi et al., 2002b).
We used TMHMM Server v. 2.0 (Denmark) for
prediction of transmembrane helices in proteins
and Parcoil for prediction of coiled-coil regions
(Berger et al., 1995). Similarity and identity values
for the comparison of AtCASP with human and
yeast proteins were established by BLASTP 2.2.10
analysis using BLOSUM62 matrix.
To generate an AtCASP-fluorescent protein
fusion, the cDNA of the Arabidopsis CASP (GenBank accession AY142680) was fused in frame
downstream of a fluorescent protein sequence
using unique BamHI and SacI sites and inserted
in pVKH18En6.
To generate the tyrosine mutant (residue 648),
we performed site directed mutagenesis by altering
the relevant codon to leucine.
Fluorescent fusions of AtNCASP and
AtCCASP were generated by PCR amplification
of the DNA encoding for the predicted N-terminus
domain of the protein (1–564aa) and the last 125
amino acids, respectively. The PCR products were
fused in frame downstream of a fluorescent protein
sequence using unique BamHI and SacI sites and
inserted in pVKH18En6.
All CASP fusions, including mutants, full
length plus N- and C-terminal fusions, were
generated as GFP and YFP fusions. The localization of each type of fusion was identical regardless
of the GFP variant in use.
For expression in mammalian cells, the fluorescent fusions of CASP and its mutants were amplified
with primers containing XhoI and XbaI sites for
subcloning in the expression vector pcDNA3 (Invitrogen). ST-RFP was generated as an in frame
fusion of the last 58 amino acids of a rat sialyltrans-
ferase fused to the monomeric RFP as explained in
Saint-Jore et al. (2002). The DNA encoding for this
fusion was subcloned into pcDNA3 using the XhoI
and XbaI sites of its multiple cloning site. The
plasmid encoding for the ER marker, luman (pcLuman-RFP, Lu and Misra, 2000) was generated by
recovering the coding sequences of luman, without
the stop codon, as well as the coding sequences of
the monomeric red F1 protein by PCR and then
assembling the two fragments between the BamHI
and XbaI sites of pcDNA3. The primers for amplifying luman incorporated BamHI and EcoRI sites
while the PCR product of RFP was bracketed by
EcoRI and XbaI sites.
The primer sequences used for the subcloning
and mutagenesis indicated above are available
upon request.
Expression Systems
Expression in plant cells
With the only exception of the GFP-AtCASPY648L
mutant, which has been expressed, transiently, for
this work we have used plant transformants
expressing stably and transiently all the other
AtCASP and derivative constructs. We have
obtained the same results with both systems. The
confocal images produced for this work have been
obtained from transient transformants. The cell
fractionation has been performed on leaves of
stable transformants for AtCCASP.
Generation of stable plant transformants was
achieved as described in Crofts et al. (1999), except
that the Agrobacterium strain used was GV3101
and selection for transformants was on hygromycin. Transformants were screened using fluorescence microscopy.
Four week old Nicotiana tabacum (cv Petit
Havana) greenhouse plants grown at 25 C were
used for Agrobacterium tumefaciens [strain
GV3101]-mediated transient expression (Batoko
et al., 2000). The bacterial optical density (OD600)
used for plant transformation was 0.05–0.1 for
CASP- and 0.2 for ERD2- and ST-tagged
constructs.
Transient expression in mammalian cells
African green monkey (Vero, obtained from
P. O’Hare, Marie Curie Institute, UK) cells were
transfected with plasmid DNA by a modification
of the technique described by Chen and Okayama
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(1998) . Briefly, 2.5 · 105 Vero cells in Dulbecco’s
minimum essential medium (Invitrogen) containing 10% newborn calf serum, penicillin and
streptomycin were added to each well of a six well
tissue culture plate (BD Falcon) containing quartz
coverslips (VWR). After incubating overnight the
culture medium was replaced and two hours later
5 lg of plasmid in transfection buffer (140 mM
NaCl, 25 mM N,N-bis(2hydroxyethyl)-2-aminoethanesulfonic acid, 0.75 mM Na2HPO4 and
125 mM CaCl2) were added to cells in each well
(Chen and Okayama, 1988). Sixteen hours later
the medium was replaced and the next day cells on
the coverslips were rinsed with phosphate buffered
saline (PBS) and fixed for 10 min in 1% paraformaldehyde. The coverslips were mounted on glass
slides in 50% glycerol in PBS.
Cell Fractionation and Protein Gel Blot Analysis
For cell fractionation experiments, we isolated
protoplasts from leaves of stable transformants
expressing AtCCASP or transient transformants
expressing ssNGFP-HDEL as described in Phillipson et al. (2001). Protoplasts were then fractionated into cytosol and vacuoles (S1),
microsomal contents (S2) and cell membranes (P)
(Denecke et al., 1992). Protoplasts were gently
resuspended in GFP extraction buffer (0.2 M
NaCl, 0.1 M Tris pH 7.8, 1 mM EDTA pH 8,
supplemented with 2% v/v b-mercaptoethanol
immediately before use), and incubated on ice for
10 min. Samples labeled + Triton were resuspended in GFP extraction buffer supplemented
with 0.2% v/v Triton X-100 to solubilize membranes. All samples were centrifuged for 15 min at
14,000 rpm and 4 C. The supernatant (S1) was
recovered and the pellet was resuspended by
sonication in GFP extraction buffer (with or
without 0.2% v/v Triton X-100), then centrifuged
for a further 15 min at 14,000 rpm and 4 C. The
supernatant (S2) was recovered and the pellet was
resuspended by sonication in GFP extraction
buffer as before (P). All samples were resuspended
to a 20-fold lower volume than that of the original
cell suspension.
Protein extracts were diluted 50:50 with 2X
SDS loading buffer (Crofts et al., 1999) and boiled
for 5 min. Equal volumes of all extracts were
loaded on SDS-polyacrylamide gels, transferred to
nitrocellulose membrane by electroblotting and
blocked with PBS, 0.05% Tween 20 and 1% milk
powder for 2 h. The filter was then incubated in
blocking buffer with anti-GFP serum from
rabbit (Molecular Probes) at a dilution of 1:1000
overnight. Further steps were performed as in
Crofts et al. (1999). The anti-GFP serum is known
to recognize all the GFP variants (Molecular
Probes).
Proteinase K treatment
Microsomes were isolated from transgenic protoplasts by osmotic shock in GFP extraction
buffer without b-mercaptoethanol, followed by
incubation on ice for 10 min. Microsomes were
then incubated on ice with or without 0.3 mg/ml
proteinase K for 30 min. After incubation the
samples were boiled for 10 min to inactivate the
proteinase. Extracts were diluted 50:50 with 2X
SDS loading buffer and the samples analysed by
western blot.
Tunicamycin treatment
Transgenic protoplasts were incubated in 20 lg/ml
tunicamycin for 24 h before extraction by sonication in GFP extraction buffer supplemented with
0.2% v/v Triton X-100. Extracts were diluted
50:50 with 2X SDS loading buffer and the samples
analysed by western blot.
Sampling and Imaging
Transformed leaves were analysed 48 h after
infection of the lower epidermis. Confocal imaging
was performed using an upright Zeiss LSM 510
META confocal microscope and a 63 · water
immersion objective. For imaging expression of
GFP constructs, YFP constructs or both, we used
imaging settings as described in Brandizzi et al.
(2002b) with a 1–3 lm optical slice. Appropriate
controls were done to exclude the possibility
of energy transfer between fluorochromes and
cross-talk. Time-lapse scanning was acquired with
imaging system software of the microscope. Fluorescence intensity measurements (plus tracking)
and post acquisition image processing were done
with Zeiss confocal and PaintShop Pro 7.0 software, respectively.
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Results
Plants have a homologous protein to the human
golgin CASP
Four integral membrane golgins have been identified in animal cells: giantin, golgin-84, golgin-67
and CASP (Linstedt and Hauri, 1993; Bascom
et al., 1999; Jakymiw et al., 2000; Misumi et al.,
2001; Gillingham et al., 2002; Barr and Short,
2003). Their identity as membrane spanning
proteins has been ascertained experimentally for
giantin, golgin-84 and CASP (Bascom et al., 1999;
Misumi et al., 2001; Gillingham et al., 2002;
Figure 1A), while golgin-67 has been predicted to
have a membrane spanning region at the C-terminus
(Jakymiw et al., 2000). Giantin, golgin-84 and
CASP share a common topology with a cytosolic
Figure 1. The plant CASP has an overall structure similar to homologues from other species. (A) Diagrams of the human (hs)
giantin, golgin-84, CASP and Arabidopsis (At) CASP indicating the potential coiled-coils and the TMDs. All these golgins share a
common topology with an extensive coiled-coil N-terminal region and a C-terminal region. Coiled-coil regions and transmembrane
domains were established by Paircoil and TMHMM software, respectively. As predicted by the Paircoil program, the extensive
coiled-coil region occupies 65% of the entire protein. (B) Diagram of entire AtCASP (residues 1–689). The large white box indicates the coiled-coil regions which are differentiated graphically from the non-coiled-coil regions by a black line. The black box
indicates the transmembrane domain of the protein (TMD). An arrowhead points at residue 564 which delimits the AtNCASP
(residues 1–564) and AtCCASP (residues 565–689). At the bottom of the panel there is a colour coded alignment of the amino acid
composition of the transmembrane domain of CASP from different species. Note the presence of the conserved tyrosine residue
(648aa; arrowhead). (C) Diagrams of the AtCASP fluorescent constructs used in this study indicating the position of a fluorescent
protein (FP, green rectangle) with respect to the amino acid residues of AtCASP. For NGFP-AtCASP, a blue rectangle indicates
the peptide bearing a glycosylation motif.
114
N-terminus and a membrane spanning domain at
the C-terminus (Figure 1A; see also Gillingham
et al., 2002). These proteins also contain several
residues in their transmembrane domain that
are strongly conserved between different species
(Gillingham et al., 2002).
In a protein-protein Blast search (NCBI Blast)
in the Arabidopsis genome using human and yeast
CASP homologues, we have identified an Arabidopsis protein that is a homologue of the CASP of
these species (AtCASP; Figure 1; see also Gillingham et al., 2002). The putative Arabidopsis protein
shows 32% identity, 55% similarity with the
human homologue and 26% identity, 45% similarity with the yeast CASP.
AtCASP is a protein of 689 residues and a
predicted molecular mass of 80 kDa. The predicted structure of the Arabidopsis protein indicates the presence of short non-coiled-coil regions
at either end of the protein and coiled-coil domains
over most of its length (Figure 1A). The program
TMHMM (Krogh et al., 2001) predicts a transmembrane domain (TMD) between residues 643–
661 (Figure 1B). A comparison of the TMD
domain of the Arabidopsis CASP with homologues
from different species indicates the presence of
highly conserved residues among different CASP
proteins (Figure 1B, see also Gillingham et al.,
2002). Strikingly, several residues central in the
TMD are invariant across species. Furthermore, a
comparison of the putative TMD region of CASP
to those of golgin-84 and giantin from different
species reveals that some of these residues are also
conserved in both golgin 84 and giantin (Gillingham et al., 2002).
These observations on CASP structure and
the comparison with the other golgins suggest
that AtCASP may be a plant membrane protein
that shares structural properties with known
CASP of different species as well as with known
integral membrane golgins such as golgin-84 and
giantin.
and giantin of other kingdoms (Linstedt and
Hauri, 1993; Bascom et al., 1999; Jakymiw et al.,
2000; Misumi et al., 2001; Gillingham et al., 2002),
we fused AtCASP to a fluorescent protein (Figure 1C) and expressed it in tobacco leaf epidermal
cells (Figure 2). Laser confocal microscopy observations on a green fluorescent protein fusion of
AtCASP (GFP-AtCASP) indicated that the fusion
labelled punctate structures (Figure 2) which were
motile in the cell (data not shown). The observed
pattern was similar to the known distribution of
Golgi stacks in tobacco leaf epidermal cells and
BY-2 cells (Boevink et al., 1998; Nebenführ et al.,
1999).
To ensure the identity of the GFP-AtCASPlabelled punctate structures, tobacco leaf epidermal cells were co-transformed with the CASP
fusion and a known Golgi marker, the Arabidopsis
H/KDEL receptor (ERD2) fused to the yellow
fluorescent protein (ERD2-YFP, Boevink et al.,
1998; Brandizzi et al., 2002a, b; Figure 3A–C).
ERD2-YFP labelled punctate structures (Figure 3B,
arrowheads) that co-localized with those labelled
by GFP-AtCASP (Figure 3C, arrowheads). ERD2
overexpression can lead to redistribution of the
receptor to the ER in mammalian cells (Tang
et al., 1993), but there is currently no published
evidence for this in plants. However, to rule out
the possibility of interference of overexpressed
ERD2 with the distribution of GFP-AtCASP, we
analysed the subcellular localization of GFPAtCASP with another known Golgi marker,
ST-YFP (Brandizzi et al., 2002a, b). As shown in
Figure 3D–F, GFP-AtCASP localized at the Golgi
apparatus labelled by ST-YFP, confirming the
results obtained with ERD2-YFP (Figure 3A–C)
AtCASP targets the Golgi apparatus in plant
cells and the targeting motifs are encoded
in its C-terminus
Targeting to the plant Golgi apparatus
In order to ascertain whether AtCASP localizes at
the Golgi apparatus like CASP and the other
integral membrane golgins, golgin-67, golgin-84
Figure 2. When expressed alone in leaf epidermal cells, GFPAtCASP shows a punctate distribution. Bar = 5 lm.
115
Figure 3. AtCASP localizes at the Golgi apparatus in tobacco leaf epidermal cells. (A–C) A GFP fusion to AtCASP (A) targets
small punctate structures in tobacco leaf epidermal cells. (B) The known Golgi marker, ERD2-YFP (Boevink et al., 1998; Brandizzi
et al., 2002a, b), labels the Golgi apparatus (arrowheads) and the ER network (arrow). (C) Merged image of A and B, showing
colocalization of the GFP-AtCASP and ERD2-YFP exclusively at the Golgi apparatus (arrowheads). (D–F) GFP-AtCASP (D) in
co-expression with ST-YFP (E) colocalized at the Golgi apparatus highlighted by ST-YFP as shown in the merged image (F). (G)
The N-terminal domain of AtCASP fused to GFP localizes in the cytoplasm. As typical for a cytosolic GFP fluorescence (daSilva
et al., 2004), organelles are highlighted in negative contrast (arrowheads). (H–J) The C-terminal domain of AtCASP fused to YFP
targets punctate structures (H). These colocalize with ERD2-GFP (I) at the Golgi apparatus as shown in the merged image of H
and I (J, arrowheads). Scale bars in C, F, G, J = 5 lm.
on the localization of GFP-AtCASP at the Golgi
apparatus.
Together, these data indicate that AtCASP
localizes at the Golgi apparatus in plant cells, as
has been shown for the mammalian CASP homologue in human cells (Gillingham et al., 2002) and
for the other integral membrane golgins such as
giantin and golgin-84 (Linstedt and Hauri, 1993;
Bascom et al., 1999; Misumi et al., 2001).
Identification of Golgi targeting domains
of AtCASP
To identify the regions of AtCASP that target the
protein to the Golgi apparatus, we fused the DNA
encoding either the N-terminal residues (1–564aa)
or the C-terminal residues (565–689aa) to a
fluorescent protein sequence (Figure 1C), and
expressed the fusion proteins in tobacco leaf
epidermal cells (Figure 3 G–J).
The N-terminal GFP fusion (GFP-AtNCASP),
which contains the N-terminal non-coiled-coil
region and the long predicted coiled-coil region
(Figure 1C), was found to distribute in the cytoplasm (Figure 3G). This result indicates that the
N-terminus of CASP is not sufficient for targeting
AtCASP to the Golgi apparatus. In contrast, the
C-terminal
YFP
fusion
(YFP-AtCCASP;
Figure 1C) that contains the predicted noncoiled-coil C-terminal domain of CASP localized
to punctate structures (Figure 3H), as observed for
the full length fusion. Subsequent co-localization
experiments using the Golgi marker ERD2-GFP
116
confirmed that these punctate structures labelled
by YFP-AtCCASP are in fact Golgi bodies
(Figure 3I–J).
These data indicate that the C-terminus rather
than the N-terminus of AtCASP has encoded
information for entering the endomembranes and
localizing at the Golgi apparatus, as is the case for
the human CASP (Gillingham et al., 2002).
To confirm that the C-terminal sequence of
AtCASP contains the integral membrane domain
of the protein as predicted by structural analysis
software, we carried out subcellular fractionation
experiments in the presence or absence of the
detergent Triton X-100 on tobacco leaves
expressing either a YFP-AtCCASP fusion or
ssNGFP-HDEL, a soluble secreted GFP bearing
a glycosylation peptide (Batoko et al., 2000;
daSilva et al., 2005) and a retention/retrieval
HDEL signal (Figure 4A). The fusion proteins
were detected on a western blot with antibodies
c
Figure 4. AtCASP is a type II membrane protein (A) Western
blot with GFP antiserum of protoplasts expressing ssNGFPHDEL or YFP-AtCCASP and fractionated into cytosol and
vacuoles (S1), microsomal contents (S2) and cell membranes
(P). In control samples ()Triton) the signal due to YFP-AtCCASP (empty arrowhead) was found mostly in the pellet containing the cell membranes, while that due to ssNGFP-HDEL
was found mostly in the microsomal contents. In samples
treated with Triton X-100 (+Triton) the signal due to YFPAtCCASP was found mainly in the soluble phase of the cell
extracts as an obvious consequence of solubilization of the
cell membranes. Similarly, a significant proportion of the signal for ssNGFP-HDEL was found in S1 due to disruption of
the microsomal membranes. Negative control (UT, untransformed protoplasts). The YFP-AtCCASP band detected in S2
is likely due to incomplete sedimentation of the microsomes
in the cell fractionation procedure. Partial rupture of the
microsomal membranes in the absence of Triton X-100
resulted in the release of some ssNGFP-HDEL into the soluble S1 phase. (B) Western blot on proteinase K treatment of
microsomes containing YFP-AtCCASP and ssNGFP-HDEL.
Treatment with proteinase K (+) resulted in complete loss of
signal for YFP-AtCCASP compared with the sample in the
absence of proteinase K ()), whereas the ssNGFP-HDEL signal was present in both lanes, although a slight reduction in
signal intensity was observed in the presence of the proteinase, due to partial rupture of the microsomes during preparation. This indicates that the YFP portion of the AtCCASP
fusion is exposed on the external face of the microsomes. (C)
Western blot showing tunicamycin treatment of NGFP-AtCASP. No shift in molecular weight is observed for NGFPAtCASP when 20 lg/ml tunicamycin was added (+), while a
significant change can be seen for ssNGFP-HDEL.
against the fluorescent protein. Consistent with the
prediction that the plant CASP is an integral
membrane protein, YFP-AtCCASP was detected
mainly in the membrane pellet of the cell extracts
(Figure 4A AtCCASP)Triton, lane P). In contrast,
ssNGFP-HDEL was found predominantly in the
soluble fraction extracted from the microsomes
(figure 4A HDEL)Triton, lane S2). Weak signals
were detected in the S1 and P lanes, probably due
to rupturing of some of the microsomes during the
initial osmotic shock (S1) or trapping of the protein
in the membrane fraction (P). To further ascertain
the distribution of the protein fusion into membranes, we treated the cell extracts with Triton
X-100, a detergent that solubilizes the membranes.
As a result of this treatment, the AtCCASP fusion
should partition into the soluble phase of the cell
extracts. As expected, treatment with Triton X-100
shifted the YFP-AtCCASP signal to the soluble
117
phase of the cell extracts (Figure 4A AtCCASP
+ Triton, lane S1). Similarly, a redistribution of a
significant portion of ssNGFP-HDEL to the cytosolic phase was observed due to the disruption of
the microsomal membranes (Figure 4A HDEL
+ Triton, lane S1).
In order to confirm the prediction data on the
orientation of the TMD of CASP, we examined
the topology of the YFP-AtCCASP fusion by
means of proteinase K protection. As a control to
establish the integrity of the membranes, we used
the secreted form of GFP bearing the ER retention
signal HDEL, ssNGFP-HDEL. The rationale of
this experiment was that if the TMD assumed a
type II orientation, the YFP-AtCCASP signal
would be exposed on the outside of the microsomes and would therefore not be protected from
the protease. As shown in Figure 4B, the peptide
due to YFP-AtCCASP was visible in the untreated
sample signal but it was not present in the sample
treated with proteinase K. The signal of ssNGFPHDEL was only partially affected, indicating that
the microsomes were mostly intact in the experiment; the slight reduction observed is likely due to
the rupturing of some microsomes during the
initial osmotic shock (compare with Figure 4A
HDEL)Triton). This control rules out the possibility that the digestion of YFP-AtCCASP with
proteinase K was due to complete rupture of the
microsomes. This experiment indicates that the
TMD of AtCASP assumes a type II orientation.
To further demonstrate the orientation of the
TMD of AtCASP, we treated cells expressing a
fusion of GFP containing an N-glycosylation site
(NGFP; Batoko et al., 2000) to the N-terminus
of AtCASP (NGFP-AtCASP; Figure 1C) with
tunicamycin, an inhibitor of N-glycosylation
(Shamu, 1997). NGFP-AtCASP localized at the
Golgi apparatus as does GFP-AtCASP (data not
shown). The N-terminal domain of CASP possesses a glycosylation consensus sequence in addition to that on the GFP moiety, increasing the
chance that a shift in molecular weight would be
observed if this part of the protein were found
within the lumen of the secretory pathway. However, according to the predicted type II orientation
of the protein, the glycosylation sites would both
be in the cytosol and would therefore not be
glycosylated. Concomitant with this hypothesis,
Figure 4C shows that treatment with tunicamycin
has no effect on the molecular weight of NGFP-
AtCASP, while treatment of cells expressing
ssNGFP-HDEL with tunicamycin gives a significant shift in the molecular weight of the protein
synthesized during the drug treatment. This indicates that neither the NGFP moiety nor AtCASP
itself was able to be glycosylated due to the
cytosolic location of the N-terminus, while the
NGFP fused to the HDEL motif is found within
the ER lumen where proteins responsible for
glycosylation are active.
Taken together, these results show that
AtCASP is a protein of the Golgi apparatus. The
absence of a signal peptide at the N-terminus of
the protein sequence and the orientation of the
TMD with the N-terminus in the cytosol and a
C-terminus in the lumen of the Golgi apparatus
suggest that the plant CASP is likely to be a type II
membrane protein, as is the human homologue
(Gillingham et al., 2002). This is the same structure
as determined experimentally not only in the
mammalian CASP but also in other integral
membrane golgins such as golgin-84 and giantin
(Linstedt and Hauri, 1993; Bascom et al., 1999;
Misumi et al., 2001; Gillingham et al., 2002).
The Arabidopsis CASP is able to target
the mammalian Golgi apparatus
AtCASP shares a large degree of homology with
its mammalian counterpart. To further explore
the identity of AtCASP as a putative Golgi matrix
protein, we aimed to investigate whether it could
target the mammalian Golgi apparatus. To do
so, we subcloned a GFP-AtCASP fusion in a
mammalian expression vector and transiently
expressed it in monkey cells (Figure 5A). Observations carried out with a laser scanning confocal
microscope indicated that the fluorescence of
GFP-AtCASP localized at a large perinuclear
structure that resembled the mammalian Golgi
apparatus (Ward and Brandizzi, 2004). To confirm the identity of this structure, we co-transformed monkey cells with GFP-AtCASP and a
red fluorescent protein fusion of a TMD domain
and cytosolic tail of a rat sialyl transferase
(ST-RFP; Figure 5B), which is known to target
the mammalian and plant Golgi apparatus
(Munro, 1995; Saint-Jore et al., 2002). The
ST-RFP fluorescence was distributed to the Golgi
apparatus (Figure 5B) and it colocalized with that
of GFP-AtCASP (Figure 5C).
118
Figure 5. AtCASP targets the mammalian Golgi apparatus. An African green monkey cell coexpressing GFP-AtCASP (A) and
ST-RFP (B). ST-RFP is a known marker of the Golgi apparatus. (C) GFP and RFP fluorescence signals overlap, indicating that
AtCASP localizes at the mammalian Golgi apparatus. Scale bar = 5 lm.
These data indicate that the plant CASP contains domains that can efficiently target to the
mammalian Golgi and reinforce the identity of the
plant CASP as a transmembrane matrix protein.
A tyrosine residue that is crucial for Golgi
localization of CASP in mammalian cells
has no effect on the Golgi distribution
of this protein in plants
The TMD of CASP contains a tyrosine residue
which is conserved across species (Figure 1B). A
GFP fusion of the human CASP bearing mutation
of the tyrosine to leucine was found to localize at
the ER and not the Golgi apparatus in mammalian
cells (Gillingham et al., 2002). To ascertain what
role this conserved amino acid plays in AtCASP
for localization at the mammalian Golgi, we
mutated the conserved tyrosine and expressed it
as a GFP fusion in monkey cells (GFP-AtCASPY648L, Figures 1C, 6A–C). We observed that
the GFP-AtCASPY648L mutant was distributed to
the ER in these cells, as shown by co-localization
experiments with an RFP fusion to a known
mammalian ER marker, luman (Lu and Misra,
2000). These data confirm that the plant CASP has
a tyrosine motif residue in the TMD that influences the localization of the protein in mammalian
cells in a similar manner to that of the human
homologue (Gillingham et al., 2002).
To investigate whether the mechanisms of
CASP localization to the Golgi apparatus are
conserved between animals and plants, we
expressed a GFP fusion of AtCASPY648L in
tobacco leaf epidermal cells. Unlike its localization
in mammalian cells, the plant mutant was found to
label exclusively the Golgi apparatus (Figure 6
D–F) of tobacco leaf epidermal cells. These data
indicate that although plant CASP contains
domains that are conserved among kingdoms,
sorting requirements for reaching the plant Golgi
apparatus appear to be independent of the tyrosine
motif, in great contrast to the situation in mammalian cells. This suggests that ER to Golgi
transport may not rely entirely on conserved
mechanisms between plants and mammals.
Discussion
Identification of a plant Golgi matrix protein
Several different Golgi matrix proteins have been
identified and characterized in yeast and mammals
(reviewed by Barr and Short, 2003). In this study
we have demonstrated the existence of a
plant Golgi integral membrane matrix protein,
an Arabidopsis homologue of the mammalian
protein CASP (Gillingham et al., 2002). Golgi
matrix proteins are thought to be important for
maintenance of the structure of the Golgi apparatus, and may also be involved in tethering of
transport vesicles through Rab GTPases (Barr,
1999; Moyer et al., 2001; Short et al., 2001;
Valsdottir et al., 2001; Gillingham and Munro,
2003). The role of Rab GTPases is being unraveled
in plants, and current data indicate roles of these
proteins in various transport routes within the cell
(Batoko et al., 2000; Ueda et al., 2000, 2001;
Rutherford and Moore, 2002; Nahm et al., 2003;
Bolte et al., 2004; Kotzer et al., 2004; Preuss et al.,
2004). It therefore seems likely that further plant
Golgi matrix proteins may exist that could specifically interact with different Rabs, but these have
yet to be identified. The characterization of at least
one plant Golgi matrix protein suggests that,
119
Figure 6. AtCASP contains signals that are important for localization of the protein at the Golgi apparatus in mammalian cells.
(A–C) An African green monkey cell coexpressing the GFP-AtCASPY648L fusion (A) and an ER marker, luman-RFP (B). The
signal due to GFP is found in the ER as shown in the merged image in C. (D–F) Tobacco leaf epidermal cells expressing a GFPAtCASPY648L fusion (D) show that the YFP fluorescence localizes at the Golgi apparatus as indicated by a colocalization with the
Golgi marker, ERD2-YFP (E). No localization of CASP at the membranes of the ER was verified as shown by the absence of
GFP labeling at the ER network highlighted by ERD2-YFP (arrowhead). (F) Merged image of D and E. Scale bars in C and
F = 5 lm.
despite the morphological differences in Golgi
organization between plants and mammals (Ward
and Brandizzi, 2004), there may be similarities
between the systems in terms of the protein
composition and function of the Golgi matrix.
Targeting of AtCASP to the Golgi has different
requirements in plants and animals
Our data indicate that AtCASP has similarities to
its mammalian homologue with regard to sequence, domain structure and intracellular localization. AtCASP is a type II transmembrane
protein, with a short luminal domain and an
extensive coiled-coil cytosolic domain that may be
involved in interactions with other proteins, or in
forming homodimers as has been shown for the
mammalian homologue (Gillingham et al., 2002).
Indeed, the homology between the plant and
mammal CASP proteins is such that AtCASP is
targeted to the Golgi in mammalian cells. However, when we mutated a conserved tyrosine
residue in the transmembrane domain that is
essential for Golgi targeting of mammalian CASP
(Gillingham et al., 2002), no effect could be
detected in the localization of the protein to the
plant Golgi apparatus. Conversely, expression
of the mutated AtCASP in mammalian cells
resulted in its distribution in the ER. We conclude
that despite the similarities between the transport
systems of plants and animals, there are also
discrepancies in the mechanisms by which some
proteins are exported to the Golgi apparatus.
The role of the tyrosine residue in mediating
the transport of CASP in mammalian cells is
unclear. Gillingham et al. (2002) reported that
mutation of the tyrosine residue does not affect the
ability of mammalian CASP to dimerize, indicating that dimerization is not a prerequisite for ER
export. It is possible that the tyrosine is involved in
interactions with other proteins that act as receptors for selecting cargo for transport to the Golgi
apparatus. If this were the case, one could postulate that homologues of these putative receptors
do not exist in plants, and that AtCASP therefore
relies on other mechanisms for export from the
ER. To gain further insights on the involvement
of specific residues that govern ER sorting in
plants, it would be useful in future to express the
mammalian CASP in a plant system.
The length of the transmembrane domain in
type I membrane-spanning proteins is important
120
in determining their destinations (Brandizzi et al.,
2002b), although no data have yet been published
to show whether a similar rule applies to type II
membrane-spanning proteins. The predicted 19amino acid transmembrane domain of AtCASP
would likely be sufficient to transport the protein
to the Golgi apparatus if type II membrane
proteins behave in the same manner as type I.
Our findings suggest that in plants the length of
the transmembrane domain may supersede a
tyrosine motif for export of proteins from the
ER. Our data do not exclude that specific signals
in the cytosolic domain of AtCASP may be
responsible for mediating its transport to the
Golgi apparatus. Two types of export signals have
recently been identified in plant transmembrane
proteins (Contreras et al., 2004; Yuasa et al.,
2005), opening up the possibility that similar
signals may be present in many other plant
proteins. Misumi et al. (2001) have shown that
the first 100 amino acids of the cytosolic domain
adjacent to the transmembrane segment are essential for the correct targeting of several golgins in
mammals. This corresponds to our finding that the
C-terminal 125 amino acids of AtCASP (giving 78
amino acids in the cytosolic domain) are sufficient
to instigate transport of the protein to the Golgi
apparatus. Further investigation into the transport
mechanisms of type II transmembrane proteins
will be necessary in order to clarify the means by
which AtCASP travels to the plant Golgi.
Conclusions
Our findings open fascinating new avenues of
research on the role of plant matrix proteins in
maintenance of the structure of the Golgi
apparatus and on protein transport occurring in
this organelle in plant cells. Deletion of the
Saccharomyces
cerevisiae
CASP-homologue,
COY1 does not affect viability, but restores
normal growth to cells lacking the Golgi soluble
N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Gos1p (Gillingham et al.,
2002). It has also been established that CASP
contains an internal coiled-coil motif that is
required for cytohesin binding both in vitro and
in COS-1 cells (Mansour et al., 2002). The specificity of the coiled-coil of CASP is not restricted to
cytohesin, however, because it is also capable of
interacting with other members of the cytohesin/
ARNO family, ARNO and ARNO3 (Mansour
et al., 2002). The function of CASP in plant cells is
unknown but some clues are provided by its Golgi
localization, structure and sequence similarity to
its homologues in other species and other golgins
such as golgin-84 and giantin (Lindstedt and
Hauri, 1993; Bascom et al., 1999; Gillingham
et al., 2002). The exclusive localization of AtCASP
at the Golgi apparatus suggests that the protein
either leaves the ER rapidly or cycles with a slow
rate from the Golgi apparatus. Therefore,
At-CASP fusion proteins may serve as cleaner
Golgi markers because they do not exhibit the
weak ER staining observed for the other Golgi
markers ST-YFP and ERD2-YFP. This feature
may be at the basis of the involvement of this
protein in events of either vesicular tethering or
establishment and maintenance of the Golgi apparatus. The identification of the binding partners of
CASP in plant cells will shed light on the role of
this golgin in the organization, structure and
trafficking pathways of the Golgi apparatus in
plants.
Acknowledgements
We acknowledge for financial support the
University of Saskatchewan and the Department
of Biology, U of S, CFI and Canada Research
Chair (CRC) grants to F.B for the development
of this work. S.H. is supported by a CRC
Provincial Operating Fund and a Department of
Biology Post-Doctoral Award. CRC Provincial
Operating Fund and Graduate College Studies
Award are acknowledged for the support of
G.S. CRC Provincial Operating Fund supports
L.B. For her MSc studentship, L.R. is indebted
to a University of Saskatchewan New Faculty
Award.
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