Download The human apyrase-like protein LALP70 is lysosomal

2473
Journal of Cell Science 112, 2473-2484 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0257
A human intracellular apyrase-like protein, LALP70, localizes to
lysosomal/autophagic vacuoles
Annette Biederbick1, Scott Rose2 and Hans-Peter Elsässer1,*
1Department of Cell Biology, Robert-Koch Str. 5, 35033 Marburg, Germany
2UT Southwestern Medical Center, Department of Molecular Biology and Oncology,
5323 Harry Hines Blvd, Dallas, TX 75235-
9140, USA
*Author for correspondence (e-mail: [email protected])
Accepted 26 May; published on WWW 7 July 1999
SUMMARY
Using antibodies against autophagic vacuole membrane
proteins we identified a human cDNA with an open reading
frame of 1848 bp, encoding a protein of 70 kDa, which we
named lysosomal apyrase-like protein of 70 kDa (LALP70).
Sequence analysis revealed that LALP70 belongs to the
apyrase or GDA1/CD39 family and is almost identical to a
human uridine diphosphatase, with the exception of nine
extra amino acids in LALP70. Members of this family were
originally described as ectoenzymes, with some
intracellular exceptions. Transfected LALP70 fused to
the green fluorescent protein localized in the cytoplasm
with a punctate pattern in the perinuclear space. These
structures colocalized with the autophagic marker
monodansylcadaverine and the lysosomal protein lamp1.
Hydrophobicity analysis of the encoded protein revealed a
transmembrane region at the N and C termini. Most of the
sequence is arranged between these transmembrane
domains, and contains four apyrase conserved regions.
In vitro transcription/translation in the presence of
microsomes showed that no signal sequence is cleaved off
and that the translation product is protected from trypsin
treatment. Our data indicate that LALP70 is a type III
lysosomal/autophagic vacuole membrane protein with the
apyrase conserved regions facing the luminal space of the
vacuoles.
INTRODUCTION
autophagic vacuoles are myelin-like membrane whirls filling
the lumen of these organelles (Seglen, 1987; Papadopoulos and
Pfeiffer, 1987). This membrane material is thought to represent
remnants from degraded organelles. However, it cannot be
ruled out that parts of the plasma membrane are also present
in autophagic vacuoles, since a connection between the
endocytic and autophagic pathway has been demonstrated
(Tooze et al., 1990; Punnonen et al., 1993). It has recently been
shown that autophagic vacuoles can be stained with the
autofluorescent substance monodansylcadaverine (MDC;
Biederbick et al., 1995), due to an interaction of MDC with the
highly concentrated lipids in these organelles (A. Niemann et
al., unpublished).
Studies on the structure, function and turnover of autophagic
vacuoles in higher eukaryotic cells have been mostly
descriptive. However, in yeast remarkable progress has been
achieved in describing the mechanism of autophagy on the
molecular level. An autophagy-like process in yeast can be
induced by starving the cells in culture and it is assumed that
the subsequent delivery of cytoplasmic domains to the vacuole
is at least in part comparable to autophagy in higher eukaryotes
(Takeshige et al., 1992; Baba et al., 1994). Applying a genetic
approach, a variety of mutants defective in autophagy have been
described: apg 1-15 (Tsukada and Ohsumi, 1993), aut 1-8
Macroautophagy is a widespread phenomenon in the
degradation of cellular components in the lysosomal
compartment (Dunn, 1994). The structural correlate in
macroautophagy is the autophagic vacuole, the formation of
which has been subdivided into two consecutive steps:
formation of autophagosomes, which lack lysosomal
hydrolases and are delineated by two membranes, and their
subsequent development into autophagolysosomes, which
contain lysosomal hydrolases and are delineated by a single
membrane (Dunn, 1990a,b; Yokota, 1993; Yokota et al., 1993).
Autophagosomes develop from endomembranes, most likely
from ribosome-free cisternae of the endoplasmic reticulum
(Furuno et al., 1990), which sequester cytoplasmic domains or
intracellular organelles addressed for degradation (Dunn,
1990a). In a subsequent step the endomembranes fuse and
constitute a closed vacuole lined by a double membrane (Dunn,
1990b).
The crucial event in the transition from an autophagosome
to an autophagolysosome is the fusion with lysosomes.
Classification of vacuoles as mature autophagic vacuoles is
predominantly based on their appearance in the electron
microscope. The ultrastructural hallmarks of fully developed
Key words: Apyrase, Lysosome, Autophagy, Membrane protein
2474 A. Biederbick, S. Rose and H.-P. Elsässer
(Thumm et al., 1994) and Tor (Noda and Ohsumi, 1998). While
only a few of the mutated genes have been cloned so far, the
function of the identified genes has only been partly unraveled.
It has been shown that apg1/aut3 are identical proteins and
represent a Ser/Thr kinase (Straub et al., 1997), the function or
substrates of which are not known. Tor has been described as a
phosphatidylinositol kinase acting upstream of the apg mutants
(Noda and Ohsumi, 1998). Interestingly, in Tor mutants
autophagy is also induced in yeast growing in nutrient-rich
medium, indicating that Tor negatively regulates autophagy.
Finally, in a recent report the conjugation of apg 12 with apg 5
has been described, with apg 7 transiently conjugated to apg 12
as an activator necessary for the subsequent apg 5 conjugation
(Mizushima et al., 1998). This model has striking similarities
to the ubiquitin system, with apg 12 as an analog of ubiquitin
and apg 7 as an analog to the ubiquitin activating enzyme E1
(Varshavsky, 1997). Indeed, apg 7 contains sequence homology
to the yeast E1 analog Uba1.
Where all these mutants are involved in the regulation of the
start of autophagy, other genes are critical for the delivery of
autophagocytosed material to the vacuole. Aut2 and aut7 have
been described as constituents of a protein complex responsible
for a proper association between autophagic vacuoles and
microtubules (Lang et al., 1998), indicating that a microtubuledependent transport of autophagic vacuoles is necessary for
their delivery to the yeast vacuole.
Although the genetic approach in yeast has been proved an
effective model to dissect the molecular basis of a variety of
cellular structures and functions, higher eukaryotic cells are
often more complex. Moreover, although there are striking
similarities between autophagy in yeast and in higher
eukaryotic cells, it is still a matter of debate how congruent this
process is in these different cell types. In order to analyse the
structure and function of autophagic vacuoles in higher
eukaryotes, we isolated autophagic vacuole membrane proteins
and used them as antigens to generate polyclonal antibodies.
We used these antibodies to screen an expression library derived
from human pancreatic adenocarcinoma cells. We obtained one
cDNA encoding a protein homologous to the members of the
dinucleotide phosphatase (apyrase, E.C. 3.6.1.15) family.
Apyrases are enzymes that cleave triphosphate nucleotides or
diphosphate nucleotides to monophosphate nucleotides. With
exceptions in yeast (Abeijon et al., 1993), Toxoplasma gondii
(Bermudes et al., 1994), potato (Handa and Guidotti, 1996) and
human (Wang and Guidotti, 1998), apyrases belong to the
family of ecto-ATPases, which are located in the plasma
membrane exposing their enzymatic activity to the extracellular
space (extensively reviewed by Plesner, 1995). Here we
describe the human intracellular apyrase-like protein LALP70,
which is associated with the autophagic/lysosomal
compartment. We provide further evidence that LALP70 is a
membrane protein exposing its functional domain to the luminal
space of autophagic vacuoles/lysosomes. A possible role of this
enzyme in nucleotide metabolism is discussed.
MATERIALS AND METHODS
Cell lines and culture conditions
The cell line PaTu 8902 was established from a human primary
pancreatic adenocarcinoma and characterized in detail as described
previously (Elsässer, 1993). Cells were cultivated in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 2 g/l Hepes, 5%
fetal calf serum, 5% adult calf serum, 50 µg/ml gentamicin (Gibco,
Karlsruhe, Germany) and incubated at 37°C in a humidified chamber
equilibrated with 5% CO2.
Antibodies
The polyclonal AV1-serum was raised against a mixture of
purified membrane proteins from autophagic vacuoles. Isolation
of autophagic vacuoles by subcellular fractionation from PaTu
8902 cells was described elsewhere (Biederbick, 1995). For one
preparation cells from 6×55 cm2 plates were used. The intact
organelles were biotinylated with the membrane-impermeable
sulfonitrohydroxysuccinimidobiotin (Pierce Chemical Corp., USA)
according to the cell surface biotinylation techniques of Zurzolo et al.
(1994). In brief, organelles were washed twice with ice-cold PBS-CM
[PBS, pH 7.2, supplemented with 1 mM MgCl2, 0.1 mM CaCl2, 1
µg/ml Leupeptin, 1 µg/ml Pepstatin A (Sigma, Deisenhofen,
Germany) and 5 µl/ml Trasylol (Bayer, Leverkusen, Germany)], and
incubated on ice for at least 60 minutes with 0.25 mg/ml Sulfo-NHSBiotin in PBS-CM in a final volume of 1 ml. The reaction was stopped
adding NH4Cl at a final concentration of 50 mM. After 10 minutes on
ice vacuoles were centrifuged at 4°C at 13,000 g for 30 minutes. The
pellet was resuspended in 250 µl lysis buffer (20 mM Tris/HCl, pH
5.0, 150 mM NaCl, 1% Triton X-100 and proteinase inhibitors as
described above) and free biotin was removed with a Fast Desalting
column HR 10/10 equipped with Sephadex G-25 Superfine
(Pharmacia, Uppsala, Sweden). The biotinylated membrane proteins
were incubated overnight at 4°C with streptavidin-agarose beads
(Sigma Chemical Co., USA) equilibrated in lysis buffer. The agarose
beads with the bound biotinylated autophagic vacuole membrane
proteins were washed twice with PBS-CM supplemented with
proteinase inhibitors and resuspended in 100 µl PBS-CM. Material
from six preparations was pooled. 200 µl were mixed with 300 µl
Gerbu adjuvans (Gerbu Biotechnik, Germany) and injected
subcutaneously into rabbits to raise polyclonal antibodies against the
membrane proteins.
Other antibodies used for immunofluorescence microscopy were a
mouse monoclonal antibody against purified anti-human CD107a
(LAMP-1) (Pharmingen, Germany), a Cy2-labelled goat anti-rabbit
IgG polyclonal antibody and a Cy3-labelled goat anti-mouse IgG
antibody (both from Amersham Pharmacia Biotech Europe GmbH,
Freiburg, Germany).
cDNA expression library and cloning
AV1-serum was used for immunoscreening a Uni-ZAP XR cDNA
Library (Stratagene, LJ, USA) derived from human pancreatic
adenocarcinoma cell line CF Pac-1. AV-1 serum was cleared from
antibodies against bacterial antigens by the method of Gruber and
Zingales (1995). Immunoscreening was performed according to the
manufacturer’s instructions (picoBlue Immunoscreening Kit,
Stratagene, USA). Three rounds of expression and immunoscreening
yielded a single clone insert of 2.4 kb in a pBluescript SK vector. The
insert was sequenced on both strands using the Perkin Elmer Applied
Biosystems 377 DNA Sequencer (Applied Biosystems, USA). The
nucleotide and deduced protein sequences were screened against the
GenBank/EMBL database using the FASTA/BLAST programs as
implemented on the internet resource. Sequence alignments were
performed using the CLUSTAL W (1.74) Multiple Sequence
Alignment software as implemented on the internet resource by the
ExPasy server.
Expression plasmids
A 1960 bp SmaI fragment from the LALP70 cDNA clone, containing
the full-length cDNA without the stop codon and the last three
predicted amino acids, was cloned in-frame into the SmaI site of the
mamalian expression vector pEGFP-N3 (Clontech Laboratories Inc.,
The human apyrase-like protein LALP70 is lysosomal 2475
USA), with EGFP fused to the C terminus of LALP70. This construct
was used to express LALP70/EGFP under the control of the
cytomegalovirus (CMV) promoter. The correctness of the reading
frame after cloning was controlled by sequencing both ligation sites.
The pVA RNAI plasmid was used as described by Svensson and
Akusjärvi (1984) and was kindly provided by R. E. Hammer, UT
Southwestern Medical Center, Dallas, Texas. The noncoding VA
RNAI was shown to be a translational enhancer (Svensson and
Akusjärvi, 1984) and has been proved to stimulate translation in
transient expression assays (Akusjärvi et al., 1987).
Transient transfections
PaTu 8902 cells were transiently transfected either with pEGFP or
pLALP70/EGFP, respectively, using a low molecular weight
polyethyleneimine (LMW-PEI), kindly provided by Th. Kissel, Dept.
of Pharmaceutical Technology, University of Marburg. Briefly, 4×105
cells were plated in 21 cm2 dishes and cultured for 24 hours. Then
cells were covered with 2.5 ml fresh DMEM containing 5% FCS and
5% HS. 9 mg of LMW-PEI was dissolved in 10 ml aqua bidest and
sterile-filtered. 100 µl of the PEI-solution were mixed with 650 µl of
150 mM NaCl and 10 µg plasmid DNA were mixed with 750 µl of
150 mM NaCl (for cotransfection experiments 10 µg from each
plasmid DNA were used). Both solutions were incubated for 10
minutes at room temperature. The LMW-PEI solution was added
dropwise to the DNA solution and this mixture was further incubated
for 10 minutes at room temperature. 500 µl were added to the medium
of one dish, and cells were incubated at 37°C in a humidified chamber
for 6 hours. The incubation medium was replaced by new DMEM
supplemented with serum and cells were further cultured for 12-42
hours.
Immunofluorescence
PaTu 8902 cells were plated on 8-well plastic LabTeks (Nunc,
Hamburg/Germany) and either further processed for transfection
according to the protocol described above or cultured for 36 hours.
Cells were fixed for 30 minutes in 4% paraformaldehyde, and
aldehyde fixation was quenched with 50 mM NH4CL in PBS for 10
minutes. Cells were permeabilized by a 5-minute incubation in 0.1%
Triton X-100, washed three times with PBS and blocked with 3%
BSA/PBS for 30 minutes. AV1-serum (1:100 in BSA/PBS) and antiCD 107a (1:1000 in BSA/PBS) were incubated overnight at 4°C.
After washing with PBS cells were further incubated with speciesspecific anti-IgG antibodies conjugated to Cy2 or Cy3 (1:1000 in
BSA/PBS), respectively, for 60 minutes. Cells were mounted with
mowiol (Hoechst, Frankfurt, Germany). For staining of autophagic
vacuoles a monodansylcadaverine (MDC) stock solution (0.1 M in
DMSO) was diluted 1:1000 in medium and applied to the cells for 60
minutes. Cells were extensively washed with PBS, fixed with 4%
paraformaldehyde and further processed for immunostaining as
described above.
Fluorescence microscopy
Cells stained with MDC or MDC and other fluorochromes were
analysed using a Dialux fluorescence microscope (Leitz, Heidelberg,
Germany) equipped with a filter system A (excitation wave length,
340-380 nm; barrier filter, 430 nm) and a filter system N2 (excitation
wave length, 530-560 nm; barrier filter, 580 nm). For detection of
LALP70-EGFP or EGFP in living or paraformaldehyde- fixed cells a
confocal laser scanning microscope LSM 410 (Zeiss, Köln,
Germany), equipped with the microscope Axiovert 135, was used.
Living cells were incubated on a thermostated and CO2-perfused stage
that was kept at 37°C during analysis. Excitation illumination was
from an argon laser (488 nm), and fluorescence was detected using a
515 nm filter. When Cy3-conjugated secondary antibodies were used
in double-staining experiments on fixed cells, excitation illumination
was from an HeNe laser (543 nm), and fluorescence was detected
using a 570 nm filter. Images were obtained using a ×40/1.3 oil-
immersion objective and pictures were taken on an APX 100 film
(AGFA, Köln, Germany) using an imagerecorder (Focus Graphics,
Foster City, USA).
In vitro transcription/translation
A SacI/XhoI LALP70 cDNA fragment containing the full-length
coding region was subcloned into the SacI/SalI sites of the pGEM4Z
vector, providing a SP6 RNA polymerase transcription initiation site
(Promega, Madison, USA). In vitro transcription/translation was
performed using the TNT-coupled reticulocyte lysate system
(Promega) using [35S]methionine (ICN Biomedicals GmbH,
Eschwege, Germany) according to the manufacturer’s instructions.
Translational processing events were analyzed by in vitro translation
in the presence or absence of canine pancreatic microsomal
membranes (Promega), combined with or without a subsequent
trypsin digestion (0.1 mg/ml trypsin for 30 minutes on ice) in the
presence or absence of 0.1% Triton X-100. Samples were separated
by 10% SDS-PAGE and gels were then treated with Entensify NEF992G (DUPONT, Brussel/Belgium), dried and exposed to Kodak XOmat X-ray film at −80°C.
Northern hybridization
A human RNA master blot (Clontech Laboratories, Inc., Palo Alto,
CA) containing 89-514 ng of each poly(A)+RNA per dot from 50
different human tissues was hybridized with a 672 bp HindIII
fragment from the LALP70 cDNA clone. RNA from PaTu 8902 cells
were isolated using RNA-clean (AGS, Heidelberg, Germany). For
northern blot analysis 15 µg total RNA were run on a formaldehydecontaining agarose gel, transferred to nitrocellulose by capillary
blotting according to standard procedures (Sambrook et al., 1989) and
hybridized with the same fragment, as described above. The
hybridisation probe was labeled with [γ-32P]dATP by random priming
using the multiprime DNA labelling systems according to the
manufacturer’s protocol (Amersham Int., UK). Hybridization was
performed at 65°C in ExpressHyb hybridization solution (Clontech
Laboratories Inc), washed according to the manufacturer’s protocols
and autoradiographed on Kodak X-Omat AR film at −78°C or,
alternatively, exposed to a PhosphorImager Screen and analyzed using
ImageQuant software (Raytest, Germany).
RESULTS
Isolation and characterization of the LALP70 cDNA
clone
Autophagic vacuoles are characterized by myelin-like
membrane whirls filling the lumen of these organelles. In order
to obtain an antiserum against membrane proteins located in
the membrane delineating the autophagic vacuole, we isolated
these organelles from human pancreatic adenocarcinoma cells
as MDC-positive vacuoles with a density of about 1.098 g/cm3
(Biederbick et al., 1995). The protein residues exposed on the
outer membrane were biotinylated and separated from proteins
of the inner membranes with streptavidin-agarose beads.
Proteins enriched by this procedure were used to raise a
polyclonal antiserum (AV1). When AV1 was used for
immunofluorescence staining of PaTu 8902 cells, a perinuclear
and punctate pattern was observed, reminiscent of the
structures stained by MDC (Fig. 1A). Double staining using an
antibody against the lysosomal membrane protein lamp1
revealed that AV1-positive and lamp1-positive structures were
mainly colocalized (Fig. 1A,B). Furthermore, lamp1-positive
organelles were also mainly colocalized with MDC-positive
structures (Fig. 1C,D), indicating that AV1 recognizes
lysosomal/autophagic vacuoles.
2476 A. Biederbick, S. Rose and H.-P. Elsässer
Fig. 1. Double fluorescence
staining of PaTu 8902 cells
using either the antiserum AV1
against autophagic vacuole
membrane proteins (A), an
antibody against the lysosomal
membrane protein lamp1 (B,D)
or the autophagic vacuole
marker monodansylcadaverine
(MDC; C). Pictures in A and B
are taken with a confocal
microscope from the same cells,
pictures in C and D are taken
with a conventional microscope
from the same cells. Note that
AV1 is colocalized with lamp1
(A,B), and that lamp1 is
colocalized with MDC
(arrowheads in C,D), although
the fluorescence intensity
differs between corresponding
granular structures when MDC
and lamp1 are compared
(arrows). Bar, 10 µm.
AV1 serum was used to screen a commercially available
human expression library derived from pancreatic
adenocarcinoma cells. After three rounds of screening the
isolated clones were sequenced and we obtained eight
independent clones. One contained a 2.4 kb insert, which was
fully sequenced from both the 3′ and 5′ directions, revealing
an open reading frame between nucleotides 170 and 2018. The
amino acid sequence deduced from this cDNA is a 616-aminoacid protein (Fig. 2). We designated this protein LALP70.
Sequence analysis
The methionine codon in position 170 is the first ATG in the
sequence and conforms to the consensus eukaryotic
translation sequence (Kozak, 1989) as a strong initiator codon
with the purines A at position −3 and G at position +4 of the
coding sequence (Fig. 2). Sequence comparison using the
BLASTN program revealed high sequence similarity to the
human mRNA for a human uridine diphosphatase located in
the Golgi apparatus (Wang and Guidotti, 1998; accession
number AF016032). Comparing with the uridine
diphosphatase sequence, the LALP70 sequence had an extra
stretch of 24 bp (1028-1052 bp) encoding eight amino acids.
Furthermore, the uridine diphosphatase sequence had an extra
triplet between 299 and 300 bp of the LALP70 sequence. In
particular the additional eight amino acids in the LALP70
sequence indicate that there are possibly several tissuespecific isoforms of LALP70, because the uridine
diphosphatase cDNA was isolated from a human brain
expression library and the LALP70 cDNA shown here was
isolated by screening a pancreas cDNA library. The encoded
protein had a calculated molecular mass of 70255 Da and a
theoretical pI of 8.55. Analysis of the deduced amino acid
sequence using BLASTP and multiple sequence alignment
indicates that the LALP70 protein is almost identical (see
above) to the Golgi-located human uridine diphosphatase
(AF016032), and homologous to the human apyrase CD39 as
well as to other 13 known apyrases from different species,
including plants (human CD 39, P49961; mouse CD39,
P55772; mouse ecto-ATPase, AF042811; human CD39-like
1 gene, U91510; human brain ecto-apyrase, AF034840; rat
brain ecto-ATPase, Y11835; Drosophila apyrase, AF041048;
yeast GDPase, P32621; C. elegans hypothetical 63 kDa
protein, Q21815; chicken ATPase, U74467; pea NTPase,
P52914; potato ATPase, P80595; yeast hypothetical 71 kDa
protein, P40009). Although the overall similarity to the
homolog sequences is only 19-29%, the relatedness to the
apyrase family is evident in four sequence clusters in the Nterminal half (boxed regions in Fig. 3), which have been
described by Handa and Guidotti (1996) as apyrase conserved
regions (ACR). Amino acid alignments of the LALP70 and
hUDPase (Wang and Guidotti, 1998) amino acid sequences
with the predicted sequences of the other known human
members of this family, CD39 (Maliszewski et al., 1994),
CD39-like genes (Chadwick and Frischauf, 1997, 1998), and
The human apyrase-like protein LALP70 is lysosomal 2477
a brain ecto-apyrase identical with CD39L3 (Smith and
Kirley, 1998), show overall similarities of 20-23%, with
slightly higher homologies in the N-terminal half (23-28%)
Fig. 2. Nucleotide sequence of the
LALP70 cDNA and the deduced amino
acid sequence of the open reading
frame. The open reading frame (start
and stop codons in bold letters) is
flanked by 169 bp of 5′ untranslated
region (5′-UTR) and 311 bp of 3′-UTR.
Amino acid sequences of the two
transmembrane domains are underlined
(see Fig. 4), the N-terminal membraneassociated domain is marked with a
dotted line. Putative amino acids for Nglycosylation are in bold letters. The 24
nucleotides and their corresponding
eight amino acids, which are missing in
the hUDPase sequence (see text for
detail), are in underlined bold letters.
The GenBank accession number of the
LALP70 nucleotide sequence is
AJ131358.
1
56
113
170
1
227
20
284
39
341
58
398
77
455
96
512
115
569
134
626
153
683
172
740
191
797
210
854
229
911
248
968
267
1025
286
1082
305
1139
324
1196
343
1253
362
1310
381
1367
400
1424
419
1481
438
1538
457
1595
476
1652
495
1709
514
1766
533
1823
552
1880
571
1937
590
1994
609
2051
2108
2165
2222
2279
G
CCC
AAT
ATG
M
CCA
P
GTC
V
TAT
Y
ATT
I
GGT
G
CAT
H
AAA
K
ATT
I
ACA
T
AAA
K
TCT
S
GGC
G
GTG
V
GCG
A
ACT
T
TTT
F
ACG
T
GCC
A
ATG
M
GGA
G
CAG
Q
CCC
P
ACC
T
GCA
A
CTG
L
TGG
W
AAG
K
TAC
Y
CAC
H
TTC
F
AGG
R
GCC
A
GGA
TTC
TCA
AGA
AAT
CAC
GGC
TGT
GGG
G
GTA
V
CTG
L
GGG
G
GAA
E
AGC
S
GAT
D
ATA
I
TCT
S
CCT
P
GCT
A
GAC
D
ATT
I
GAA
E
GGC
G
GTA
V
AAC
N
TTT
F
AAC
N
CCG
P
CAA
Q
CCT
P
CCA
P
GAG
E
AAG
K
TAC
Y
ATG
M
ACT
T
AGG
R
ACC
T
CTG
L
CGC
R
CAG
Q
AAA
TTT
CGC
TCG
TAG
GAG
CGG
TTG
AGG
R
GGG
G
GCT
A
CGA
R
GCT
A
AGT
S
CTG
L
AAA
K
CCA
P
CTC
L
ATT
I
TCT
S
AAT
N
GTT
V
ATT
I
AGC
S
TTG
L
CTT
L
ACC
T
TAC
Y
ACC
T
TTC
F
ATT
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GAT
D
GAT
D
GCC
A
TTT
F
GCC
A
ACC
T
CAC
H
GTG
V
ACT
T
AAT
N
GCC
CCC
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AGA
CTG
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ATT
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ACA
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CCG
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CTT
L
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CAT
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CTC
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GGA
G
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ATT
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L
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CAC
H
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TAT
Y
TCT
S
GAG
E
TTG
L
CGC
R
TGG
W
GTG
V
CCC
P
GCC
A
ATT
TTT
TAA
CCA
GGT
versus the C-terminal half (17-19%) similarities (Table 1).
Hydropathy analysis of the protein using the Kyte-Doolittle
algorithm is shown in Fig. 4 and reveals that there are two
GCT
AGC
GAA
GGC
G
CCT
P
GCT
A
ACC
T
GAC
D
TCT
S
GAT
D
GGC
G
TTG
L
ATT
I
GAA
E
GCA
A
GTC
V
ATT
I
GAC
D
GCG
A
T GT
C
TTT
F
CAA
Q
GAC
D
TAC
Y
AAT
N
TTC
F
TTA
L
TGT
C
CAT
H
GTG
V
CAA
Q
TTT
F
CGG
R
CTG
L
CGG
R
CCG
P
TTT
TTT
TCC
TCC
GTG
GGC
AGG
GGA
ATC
I
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R
GTT
V
AGA
R
ACC
T
CGA
R
ATC
I
ATT
I
AAC
N
CTC
L
GAC
D
GAA
E
CTT
L
CCT
P
ATG
M
TCC
S
GAT
D
GGT
G
AAG
K
CCC
P
CTA
L
AAA
K
CAG
Q
CGA
R
GCA
A
GCT
A
TTT
F
GTT
V
CTA
L
GGC
G
CTG
L
AGC
S
GGG
G
GCC
GTT
CAG
TGG
GTG
CGT
AGG
CTG
TCC
S
ATT
I
TCA
S
GAC
D
AAT
N
GTA
V
AGG
R
TCA
S
TTT
F
TGC
C
CTT
L
GTA
V
GGA
G
GGA
G
GGC
G
TCA
S
GTT
V
GGC
G
AAC
N
TGC
C
CGA
R
ACA
T
AAC
N
ATG
M
ACA
T
GAC
D
CAT
H
TAC
Y
CCG
P
GTT
V
GCC
A
AGC
S
ACC
T
TCA
CCT
CAC
CTA
GCG
GAT
GCT
AAT
TGT
C
CTG
L
CTT
L
AAG
K
AAC
N
TTT
F
CAA
Q
GAA
E
GCT
A
ACG
T
CTG
L
ATT
I
CGA
R
AGT
S
GGC
G
CAG
Q
CAC
H
AAT
N
AGG
R
CTA
L
GGG
G
AAC
N
AGT
S
GGG
G
AAG
K
CTC
L
AGG
R
GAC
D
TTA
L
TCC
S
ATC
I
TCG
S
TTG
L
GGG
CCA
TTT
ACA
GGC
GCT
CTA
CCC
CTT
L
AAT
N
TTA
L
AAA
K
CCC
P
GTT
V
ATG
M
TTT
F
GCA
A
GCT
A
ACC
T
TCT
S
TTT
F
GAA
E
GTG
V
CAG
Q
CAA
Q
GCT
A
CTC
L
CCC
P
ACT
T
GAG
E
GAA
E
GGA
G
TGG
W
CAC
H
GGC
G
AAG
K
AGA
R
TTT
F
CTG
L
GCC
A
TGA
*
TTT
AAC
GGG
CGG
GCC
GCC
ACT
AGA
TTT
F
ACC
T
TAT
Y
TTT
F
AAT
N
TAC
Y
AGG
R
GCT
A
GAG
E
GGA
G
GAT
D
GGG
G
GAG
E
AGC
S
TCG
S
GAA
E
ACT
T
GCT
A
CTG
L
CTA
L
GGA
G
ACC
T
TTC
F
GAC
D
TCC
S
AGG
R
TTT
F
GAG
E
GAC
D
GTC
V
CTG
L
GCC
A
TCC
CAC
CCG
GCA
CCT
P
AAT
N
TTT
F
CAA
Q
GTG
V
TGC
C
GAT
D
ACC
T
CAT
H
ATG
M
ATC
I
AAA
K
CAT
H
AGC
S
ACT
T
GAA
E
GAG
E
CGA
R
GGT
G
GAC
D
GAC
D
CAG
Q
TAT
Y
TAC
Y
ATT
I
CTT
L
TCG
S
GTT
V
ATC
I
TAC
Y
TAC
Y
GCC
A
AGC
TGG
TGA
TTG
GCT
A
TTA
L
TCT
S
AGG
R
AAC
N
TGG
W
AAA
K
TCT
S
GTG
V
AGA
R
CCC
P
CAG
Q
ATT
I
GAA
E
CAG
Q
GTA
V
CAT
H
CAG
Q
AAA
K
AT T
I
TTT
F
ACT
T
GGC
G
AAT
N
TTG
L
AAG
K
TTT
F
CAG
Q
CAG
Q
AAC
N
CTG
L
CTC
L
TCA
TGG
GGA
CCT
TCT
S
CGC
R
GTT
V
TAC
Y
TAT
Y
CCA
P
AAC
N
CCA
P
CCA
P
ATC
I
GTG
V
GAA
E
GAA
E
GCC
A
ATA
I
GCT
A
GTG
V
AGA
R
CAG
Q
AAA
K
GAC
D
TCC
S
TTC
F
GCT
A
CGG
R
TAT
Y
CCT
P
TGG
W
CAG
Q
CAC
H
CTG
L
TGG
W
CAG
TCC
CCC
TGT
TGG
W
CAA
Q
GTC
V
CTG
L
GGG
G
AGG
R
CGA
R
GAG
E
CGG
R
CTC
L
CAC
H
GGT
G
GAT
D
ATT
I
GCG
A
AAA
K
TAT
Y
TAC
Y
ACT
T
GAT
D
CTG
L
CTC
L
TCC
S
GCT
A
GAA
E
CAG
Q
GTC
V
ACC
T
GAG
E
TAC
Y
CGG
R
ATG
M
CTC
CCC
CAG
TGC
CAT
H
ATT
I
ATA
I
GCA
A
ATC
I
CAT
H
AAG
K
AAA
K
GCA
A
CCC
P
TTT
F
GTG
V
GAT
D
GTC
V
TAC
Y
AAC
N
CGA
R
GAA
E
GGT
G
GAA
E
TGT
C
AAT
N
GAA
E
AAA
K
CGC
R
TGC
C
AAC
N
CTT
L
GCC
A
CTG
L
CTG
L
GAG
E
CAC
GCT
CAT
TGA
TTT
F
ATG
M
ATC
I
CGA
R
GTG
V
AAT
N
CCA
P
GTC
V
AAA
K
GAA
E
GAC
D
TAT
Y
GAT
D
CGT
R
GAA
E
TTG
L
GTC
V
GAC
D
CTG
L
ATC
I
CGA
R
GGG
G
TTC
F
TTT
F
TTT
F
TTC
F
TAT
Y
GGA
G
TTC
F
TTC
F
CGG
R
GAG
E
GAA
CCC
TAT
CCT
AGC
S
GTC
V
CGA
R
GTT
V
GTG
V
GGC
G
GTG
V
AGT
S
CAC
H
AGC
S
TTT
F
GCT
A
GAG
E
AAA
K
GTC
V
TTA
L
TAT
Y
AGA
R
ACT
T
CAG
Q
GAG
E
GTC
V
TAC
Y
ACT
T
GAC
D
AAA
K
AAA
K
GCC
A
CGA
R
TCT
S
CGC
R
GGC
G
GAC
GGG
TTC
TTC
ATA
I
ATT
I
AAT
N
ACC
T
GAC
D
AAT
N
GTC
V
GAT
D
AAA
K
CAG
Q
CTG
L
TGG
W
GCC
A
AGG
R
CCC
P
GCT
A
GTG
V
ATA
I
CCT
P
CAA
Q
ACT
T
TAC
Y
TAC
Y
AAA
K
CGA
R
TCG
S
AGC
S
ATC
I
GCC
A
GGC
G
ATC
I
CTT
L
TCA
GTC
TAT
AGT
TCT
S
AGT
S
AAG
K
GAC
D
TGT
C
CCA
P
ATG
M
TAC
Y
GAG
E
CAG
Q
TTT
F
ATT
I
GTT
V
ACA
T
AAA
K
GAA
E
GCC
A
TTT
F
GAT
D
AAT
N
ATC
I
CAG
Q
TGC
C
GCT
A
GGA
G
GCC
A
TTA
L
CTC
L
AGT
S
TGC
C
CAC
H
CCC
P
AAA
CTC
AAA
AGG
TGA
CGT
CTC
AAC
CCG
AAC
TAG
TTT
AAA
AGG
CCC
TCC
ATT
ATG
CGG
GTC
AAC
TTC
CAG
GCG
TCT
TAC
CTG
GCC
GAT
ACT
TCA
TGG
GAG
CAT
AAA
GGA
TTT
CGC
GAG
AAT
AGG
TGT
GGT
GTC
ACA
C
TTT
GGC
AGG
AAA
2478 A. Biederbick, S. Rose and H.-P. Elsässer
Table 1. Amino acid homologies of human apyrases
CD39
CD39-L1
CD39-L2
CD39-L4
Brain-apyrase
hUDPase
LALP70
CD39
CD39-L1
CD39-L2
CD39-L4
Brain-apyrase
Golgi-UDPase
LALP70
100
38
100
16
19
100
20
19
46
100
34
38
16
21
100
22
22
20
22
22
100
21
22
20
22
23
99
100
CD39
CD39-L1
CD39-L2
CD39-L4
Brain-apyrase
Golgi-UDPase
LALP70
100
45 // 33
100
22 // 12
26 // 14
100
26 // 15
24 // 14
45 // 47
100
42 // 27
43 // 35
23 // 13
25 // 17
100
28 // 17
27 // 18
23 // 17
25 // 19
27 // 19
100
25 // 17
27 // 18
23 // 17
25 // 19
27 // 19
98 // 100
100
A
B
Comparison of the deduced LALP70 amino acid sequence with the amino acid sequences from five other known human apyrases using Clustal W. Numbers
give the percentage of similarity.
(A) Comparison of the full length sequences.
(B) Comparison of the N-terminal and the C-terminal part of the sequences (N-terminal similarity//C-terminal similarity). The N-terminal part in the LALP70
sequence was defined as amino acids 1-296, containing the apyrase conserved regions as well as the 8-amino-acid stretch unique for LALP70 (compare with Fig. 3).
potential transmembrane regions, one near the N terminus
(amino acids 37-53) and one near the C terminus (amino acids
566-582). This has also been shown for most other apyrases
described so far, especially for the hUDPase (Wang and
Guidotti, 1998). Exceptions are a yeast GDPase with only one
transmembrane region at the N terminus (Abeijon et al.,
1993), and a potato ATPase, which is soluble (Handa and
Guidotti, 1996). A third possible membrane-associated
region at the N terminus (amino acids 3-25) is consistent with
the in vitro translation experiments in the presence of
microsomal membranes (Fig. 5) discussed below. Hence, the
hydrophobicity plot suggests that the LALP70 protein is a
type III integral membrane protein (Singer, 1990). In vitro
transcription/translation of the LALP70 cDNA produced a
single major protein chain of approximately 70 kDa when
analyzed by SDS-PAGE (Fig. 5, lane 1), in good agreement
with the predicted molecular mass of 70255 Da. Translation
in the presence of microsomal membranes did not alter the
apparent size of the protein product (Fig. 5, lane 3), indicating
that no signal peptide cleavage and no glycosylation events
had occurred.
The main and the smaller polypeptide chains synthesized
in the absence of microsomes were completely digested by
trypsin (Fig. 5, lane 2). In contrast, the 70 kDa protein
synthesized in the presence of microsomes was protected
from trypsin digestion, indicating that it was cotranslationally
translocated into the microsomal vesicles. Nevertheless,
treatment of the microsomal preparation with trypsin caused
a faint band with a slighly reduced molecular mass of about
67 kDa (Fig. 5, lane 4). These bands were only digested with
trypsin, when Triton X-100 was added (Fig. 5, lane 5). In
relation to our sequence analysis data discussed above and to
the model of apyrase integration into membranes deduced by
others, we propose that the 67 kDa band represents a LALP70
degradation product where the 33 C-terminal amino acid
residues have been proteolytically cleaved. The main part of
the protein flanked by the transmembrane domains is
orientated into the lumen of the microsomal vesicles, while
the N terminus is associated with the membrane and not
accessible to trypsin.
Subcellular distribution of LALP70
To study the subcellular localisation of the LALP70 protein we
cloned the cDNA lacking only the stop codon and the last three
amino acid codons in-frame into the mamalian expression
vector pEGFP-N1, with the green fluorescent protein (EGFP)
fused to the C terminus of LALP70. PaTu 8902 cells were
transfected with this construct to express a LALP70-EGFP
fusion protein under the control of the cytomegalovirus
promotor. As soon as 12 hours after transfection the expression
of the LALP70-EGFP fusion protein could be observed by
confocal fluorescence microscopy as a punctate pattern around
the nucleus (Fig. 6A). Control cells transfected with the
expression vector missing the LALP70 domain were uniformly
fluorescent in the cytoplasm and nuclei (Fig. 6B). LALP70EGFP fluorescence became more intense when cells were
incubated for up to 72 hours and in some of the transfected
cells a diffuse cytoplasmic background was observed.
However, under no conditions and at no time after transfection
did fluorescent staining of the plasma membrane occur. These
results indicate that the LALP70-EGFP fusion protein was
associated with endomembranes and not with the plasma
membrane. To further characterize organelles associated with
the LALP70-EGFP fusion protein, transfected PaTu 8902 cells
were fixed and immunostained with various antibodies against
known marker proteins of subcellular compartments. Analysis
with a confocal laser scanning microscope revealed a
colocalization of LALP70-EGFP with lamp1, a membrane
protein enriched in lysosomal organelles (Fig. 6C-F). However,
in all transfected cells examined (>50) there were more
LALP70-EGFP positive organelles than lamp1-labeled
organelles. Furthermore, the degree of colocalization differed
from cell to cell, with some intracellular compartments
containing only lamp1 or LALP70 or containing both,
occurring in different ratios. Some of the LALP70-EGFP
positive organelles, which were not stained by the lamp1
The human apyrase-like protein LALP70 is lysosomal 2479
antibody, resembled in their morphology the Golgi apparatus
or the rough endoplasmatic reticulum. This would be in
accordance with the findings of Wang and Guidotti (1998),
who localized the hUDPase in the Golgi complex. In addition,
when transfected cells were subsequently incubated with the
autophagic vacuole marker MDC, the LALP70/EGFP fusion
protein colocalized with MDC-positive organelles (Fig. 6G,H).
These results of transient transfection and immunolocalization
illustrate the intracellular distribution of the LALP70 protein
into lysosmal structures, comparable with the MDC-positive
ACR1
LALP70
hum-UDPase
hum-CD39
mouse-CD39
mouse-ecto
rat-ecto
hum-CD39L1
chicken-ecto
hum-brain
C.el.-hyp63
drosophila
pea-ntpase
potato-ATPase
yeast-GDPase
yeast-hyp71
KYGRLTRDKKFQRYLARVTDIEATDTNNPNVNYGIVVDCGSSGSRVFVYCWPRHNGNPHD
KYGRLTRDKKFQRYLARVTDIEATDTNNPNVNYGIVVDCGSSGSRVFVYCWPRHNGNPHD
GL---TQNKALP----------------ENVKYGIVLDAGSSHTSLYIYKWPAEKEN--D
GL---TQNKPLP----------------ENVKYGIVLDAGSSHTNLYIYKWPAEKEN--D
P----TQDVREP----------------PALKYGIVLDAGSSHTSMFVYKWPADKEN--D
P----TQDVREP----------------PALKYGIVLDAGSSHTSMFVYKWPADKEN--D
P----TRDVREP----------------PALKYGIVLDAGSSHTSMFIYKWPADKEN--D
G----SGDARGP----------------PSFKYGIVLDAGSSHTAVFIYKWPADKEN--D
TVIQIHKQEVLP----------------PGLKYGIVLDAGSSRTTVYVYQWPAEKEN--N
TS---PKVIADD----------------QERSYGVICDAGSTGTRLFVYNWISTSDS--E
NASPYLARLASKFG-------------YSKVQYAAIIDAGSTGSRVLAYKFNRSFID--N
-----KIFLKQE----------------EISSYAVVFDAGSTGSRIHVYHFNQNLD---L
PL---RRHLLSH----------------ESEHYAVIFDAGSTGSRVHVFRFDEKLG---L
GYLQDSKTEQNYPELADAVKSQTSQTCSEEHKYVIMIDAGSTGSRVHIYKFDVCTS---------MLIENT-----------------NDRFGIVIDAGSSGSRIHVFKWQDTESLLHA
: : *.**: : : : :
116
117
74
74
65
65
65
63
82
69
104
68
70
116
37
LALP70
hum-UDPase
hum-CD39
mouse-CD39
mouse-ecto
rat-ecto
hum-CD39L1
chicken-ecto
hum-brain
C.el.-hyp63
drosophila
pea-ntpase
potato-ATPase
yeast-GDPase
yeast-hyp71
LLDIRQMRDKNRKPVVM------KIKPGISEFATSPEKVSDY-ISPLLNFAAEHVPRAKH
LLDIRQMRDKNRKPVVM------KIKPGISEFATSPEKVSDY-ISPLLNFAAEHVPRAKH
TGVVHQVEECRVK------------GPGISKFVQKVNEIGIY-LTDCMERAREVIPRSQH
TGVVQQLEECQVK------------GPGISKYAQKTDEIGAY-LAECMELSTELIPTSKH
TGIVGQHSSCDVR------------GGGISSYANDPSRAGQS-LVECLEQALRDVPKDRY
TGIVGQHSSCDVQ------------GGGISSYANDPSKAGQS-LVRCLEQALRDVPRDRH
TGIVGQHSSCDVP------------GGGISSYADNPSGASQS-LVGCLEQALQDVPKERH
TGVVSEHSMCDVE------------GPGISSYSSKPPAAGKS-LEHCLSQAMRDVPKEKH
TGVVSQTFKCSVK------------GSGISSYGNNPQDVPRA-FEECMQKVKGQVPSHLH
LIQIEPVIYDNKP-VMK------KISPGLSTFGTKPAQAAEY-LRPLMELAERHIPEEKR
KLVLYEELFKERK-------------PGLSSFADNPAEGAHS-IKLLLDEARAFIPKEHW
LHIGKGVEYYNKI------------TPGLSSYANNPEQAAKS-LIPLLEQAEDVVPDDLQ
LPIGNNIEYFMAT------------EPGLSSYAEDPKAAANS-LEPLLDGAEGVVPQELQ
PPTLLDEKFDMLE-------------PGLSSFDTDSVGAANS-LDPLLKVAMNYVPIKAR
TNQDSQSILQSVPHIHQEKDWTFKLNPGLSSFEKKPQDAYKSHIKPLLDFAKNIIPESHW
*:* : .
:
:.
:*
169
170
121
121
112
112
112
110
129
121
150
115
117
162
97
LALP70
hum-UDPase
hum-CD39
mouse-CD39
mouse-ecto
rat-ecto
hum-CD39L1
chicken-ecto
hum-brain
C.el.-hyp63
drosophila
pea-ntpase
potato-ATPase
yeast-GDPase
yeast-hyp71
KETPLYILCTAGMRILPES-----QQKAILEDLLTDIPVHFDFLFSD--SHAEVISGKQE
KETPLYILCTAGMRILPES-----QQKAILEDLLTDIPVHFDFLFSD--SHAEVISGKQE
QETPVYLGATAGMRLLRME-----SEELADRVLDVVERSLSNYPFDF--QGARIITGQEE
HQTPVYLGATAGMRLLRME-----SEQSADEVLAAVSTSLKSYPFDF--QGAKIITGQEE
ASTPLYLGATAGMRLLNLT-----SPEATAKVLEAVTQTLTRYPFDF--RGARILSGQDE
ASTPLYLGATAGMRPFNLT-----SPEATARVLEAVTQTLTQYPFDF--RGARILSGQDE
AGTPLYLGATAGMRLLNLT-----NPEASTSVLMAVTHTLTQYPFDF--RGARILSGQEE
ADTPLYLGATAGMRLLTIA-----DPPSQT-CLSAVMATLKSYPFDF--GGAKILSGEEE
GSTPIHLGATAGMRLLRLQ-----NETAANEVLESIQSYFKSQPFDF--RGAQIISGQEE
PYTPVFIFATAGMRLIPDEYVLIGQKEAVLKNLRNKLPKITSMQVLK--EHIRIIEGKWE
SSTPLVLKATAGLRLLPAS-----KAENILNAVRDLFAK-SEFSVDM--DAVEIMDGTDE
PKTPVRLGATAGLRLLNGD-----ASEKILQSVRDMLSNRSTFNVQP--DAVSIIDGTQE
SETPLELGATAGLRMLKGD-----AAEKILQAVRNLVKNQSTFHSKD--QWVTILDGTQE
SCTPVAVKATAGLRLLGDA-----KSSKILSAVRDHLEKDYPFPVVEG-DGVSIMGGDEE
SSCPVFIQATAGMRLLPQD-----IQSSILDGLCQGLKHPAEFLVEDCSAQIQVIDGETE
*: : .***:* :
:
:: * *
LALP70
hum-UDPase
hum-CD39
mouse-CD39
mouse-ecto
rat-ecto
hum-CD39L1
chicken-ecto
hum-brain
C.el.-hyp63
drosophila
pea-ntpase
potato-ATPase
yeast-GDPase
yeast-hyp71
GVYAWIGINFVLGRFEHIEDDDEAVVEVNIPGSESSEAIVRKRTAGILDMGGVSTQIAYE
GVYAWIGINFVLGRFEHIEDDDEAVVEVNIPGSESSEAIVRKRTAGILDMGGVSTQIAYE
GAYGWITINYLLGKFSQKTR----------WFSIVPYETNNQETFGALDLGGASTQVTFV
GAYGWITINYLLGRFTQEQS----------WLSLIS-DSQKQETFGALDLGGASTQITFV
GVFGWVTANYLLENFIKYG-----------WVGRWI--RPRKGTLGAMDLGGASTQITFE
GVFGWVTANYLLENFIKYG-----------WVGRWI--RPRKGTLGAMDLGGASTQITFE
GVFGWVTANYLLENFIKYG-----------WVGRWF--RPRKGTLGAMDLGGASTQITFE
GVFGWITANYLLENFIKRG-----------WLGEWI--QSKKKTLGAMDFGGASTQITFE
GVYGWITANYLMGNFLEKN----------LWHMWVH--PHGVETTGALDLGGASTQISFV
GIYSWIAVNYALGKFNKTAT---------LDFPGTSPAHARQKTVGMIDMGGASAQIAFE
GIFSWFTVNFLLGRLS------------------------KTNQAAALDLGGGSTQVTFS
GSYLWVTVNYALGNLG----------------------KKYTKTVGVIDLGGGSVQMAYA
GSYMWAAINYLLGNLG----------------------KDYKSTTATIDLGGGSVQMAYA
GVFAWITTNYLLGNIGANG--------------------PKLPTAAVFDLGGGSTQIVFE
GLYGWLGLNYLYGHFNDYN-----------------PEVSDHFTFGFMDMGGASTQIAFA
* : *
*:
.:
. :*:** *.*: :
LALP70
VPKTVSFASSQQEEVAKNLLAEFNLGCDVHQTEHVYRVYVATFLGFGGNAARQRYEDRIF 342
hum-UDPase
hum-CD39
mouse-CD39
mouse-ecto
rat-ecto
hum-CD39L1
chicken-ecto
hum-brain
C.el.-hyp63
drosophila
pea-ntpase
potato-ATPase
yeast-GDPase
yeast-hyp71
VPKT--------EEVAKNLLAEFNLGCDVHQTEHVYRVYVATFLGFGGNAARQRYEDRIF
PQNQ-------TIESPDNALQFRLYGKD-------YNVYTHSFLCYGKDQALWQKLAKDI
PQNS-------TIESPENSLQFRLYGED-------YTVYTHSFLCYGKDQALWQKLAKDI
TTS--------PSEDPDNEVHLRLYGQH-------YRVYTHSFLCYGRDQVLQRLLASAL
TTS--------PSEDPGNEVHLRLYGQH-------YRVYTHSFLCYGRDQILLRLLASAL
TTS--------PAEDRASEVQLHLYGQH-------YRVYTHSFLCYGRDQVLQRLLASAL
TSD--------AIEDPKNEVMLKLYGQP-------YKVYTHSFLCYGRDQVLKRLLSKVL
AGEK-------MDLNTSDIMQVSLYGYV-------YTLYTHSFQCYGRNEAEKKFLAMLL
LPDT--------DSFSSINVENINLGCREDDSLFKYKLFVTTFLGYGVNEGIRKYEHMLL
PTDP--------DQVPVYDKYMHEVVTS---S-KKINVFTHSYLGLGLMAARHAVFTHGY
VSKK------TAKNAPKVADGDDPYIKKVVLKGIPYDLYVHSYLHFGREASRAEILKLTP
ISNE------QFAKAPQNEDGE-PYVQQKHLMSKDYNLYVHSYLNYGQLAGRAEIFKASR
PTFPIN-----EKMVDGEHKFDLKFGDE------NYTLYQFSHLGYGLKEGRNKVNSVLV
PHDS-------GEIARHRDDIATIFLRSVNGDLQKWDVFVSTWLGFGANQARRRYLAQLI
:: :
*
ACR2
Fig. 3. Multiple sequence alignment of
LALP70 amino acid sequence with 14
other known apyrase amino acid
sequences obtained with Entrez
(Accession numbers are: hUDPase,
AF016032; human CD 39, P49961;
mouse CD39, P55772; mouse ectoATPase, AF042811; human CD39-like 1
gene, U91510; human brain ecto-apyrase,
AF034840; rat brain ecto-ATPase,
Y11835; Drosophila hypothetical
apyrase, AF041048; yeast GDPase,
P32621; C. elegans hypothetical 63 kDa
protein, Q21815; chicken ATPase,
U74467; pea NTPase, P52914; potato
ATPase, P80595; yeast hypothetical 71
kDa protein, P40009). Shown is the Nterminal part between amino acids 57 and
342 of the LALP70 sequence. The four
apyrase conserved regions (ACR1-4) as
defined by Handa and Guidotti (1996) are
marked by boxes. Note that the additional
eight amino acids (287-294; underlined,
italic) of the LALP70 sequence are
unique for this protein and are missing in
all other known apyrases including the
hUDPase.
ACR3
ACR3
222
223
174
174
165
165
165
162
182
179
202
168
170
216
152
ACR4
282
283
224
223
212
212
212
209
230
230
238
206
208
256
195
335
270
269
257
257
257
254
276
282
286
260
261
305
248
2480 A. Biederbick, S. Rose and H.-P. Elsässer
sequence is almost identical to a human Golgi UDPase (Wang
and Guidotti, 1998), with the exception of an additional 24 bp
stretch (from 1028 to 1052 bp) in the LALP70 sequence.
Furthermore, the hUDPase contained an extra base pair triplet
between 299 and 300 bp of our sequence. In particular the
additional 24 bp encoding eight more amino acids raise the
possibility that LALP70 may occur in differently spliced
isoforms. Since LALP70 was cloned from a pancreatic
4
3
2
Hydropathy
*
1
0
-1
-2
-3
TM
TM
-4
1
101
201
301
401
501
601
Amino Acid Residue
Fig. 4. Hydrophobicity analysis of human LALP70. The deduced
amino acid sequence was analysed according to Kyte and Doolittle
(1982). Two predicted transmembrane domains (TM) are indicated
by bars. They are located at the N and C termini of the linear amino
acid sequence, flanking the putative extracytosolic region, which
contains the four apyrase conserved regions. *An N-terminal
hydrophobic amino acid stretch, which might also be associated with
the membrane.
organelles (Fig. 1D), which were previously shown to be
autophagic vacuoles (Biederbick et al., 1995).
Tissue distribution of LALP70 RNA
To examine the distribution of LALP70 mRNA in various
human organs, a cDNA fragment of the C-terminal half of the
LALP70 sequence with lowest homologies to other human
members of the apyrase family was hybridized to a northern
dot blot of poly(A)+ RNAs from 50 different organs. LALP70
mRNA was detected in all the human tissues including fetal
tissues, although mRNA expression levels differed up to
sixfold, with the highest expression in testis (Fig. 7A, D1) and
the lowest expression in bladder (Fig. 7A, C5). Control RNA
or DNA from yeast and E. coli (Fig. 7A, H1-H4) as well as
polyadenylic acid (Fig. 7A, H5) and human repetitive DNA
(Fig. 7A, H6) were negative.
A single mRNA of about 7.4 kb was also detected in RNA
prepared from PaTu 8902 cells (Fig. 7B). This long message
indicates the presence of a very long 3′ untranslated region,
which is not present in the LALP70 clone. However, a partial
coding sequence which had been published as an EST
sequence (KIAA0392) before (AB002390, Nagase et al.,
1997), contained a 3′-UTR of about 3.8 kb.
DISCUSSION
We have cloned a 2330 bp cDNA encoding the
lysosomal/autophagolysosomal specific protein LALP70 by
immunoscreening an expression library derived from a human
pancreatic adenocarcinoma cell line. The antibody used was
raised against the biotinylated proteins of the outer membrane
of autophagic vacuoles (AVs) and recognized subcellular
structures that were also positive for the lysosomal/autophagic
vacuole markers lamp1 or monodansylcadaverine (Fig. 1). The
Fig. 5. (A) In vitro transcription/translation assay with the LALP70
cDNA cloned in the pGEM4Z vector. Transcription was performed
using the SP6 polymerase and the products separated by SDS-PAGE.
Without microsomes the main band occurred at 70 kDa, in agreement
with the calculated molecular mass of 70.3 kDa for LALP70 deduced
from the putative amino acid sequence (lane 1). The presence of
microsomal membranes did not alter the mobility of this band,
indicating that no signal sequence is cleaved off and that LALP70 is
not or only slightly glycosylated (lane 2). When LALP70 was in
vitro translated in the absence of microsomal membranes (lane 2) or
in the presence of microsomal membranes (lane 5) and subsequently
incubated with the detergent Triton X-100, the 70 kDa band
disappeared completely. However, in vitro translation products in the
presence of microsomal membranes and without Triton X-100 (lane
4) LALP70 were not (arrowhead) or only slightly (arrow) degraded,
indicating that most of the protein molecule was trapped in the
microsomes. (B) Control experiments using mRNAs for β-lactamase
(lanes 1 and 2) to show cleavage of a signal peptide (uncleaved and
cleaved form are indicated with arrows) and α-factor (lane 3-5) to
show glycosylation reaction, which had been digested by
endoglycosidase H. Both mRNAs were supplied by the
manufacturer.
The human apyrase-like protein LALP70 is lysosomal 2481
Fig. 6. Localization of the LALP70/EGFP-fusion
protein in PaTu 8902. Cells were transiently
transfected with an expression plasmid containing
either LALP70/EGFP cDNA (A) or the cDNA for
EGFP alone (B). LALP70/EGFP was located in
perinuclear granular structures (A), while EGFP
alone was homogenously dispersed throughout the
cytoplasm and the nucleus (B). In colocalization
experiments using an antibody against lamp1 (D,F),
LALP70/EGFP occurred in granular structures which
were also positive for lamp1 (C, LALP70/EGFP; D,
lamp1). However, in some cells the amount of
LALP70/EGFP positive structures exceeded those
positive for lamp1 (E, LALP70/EGFP; F, lamp1).
LALP70/EGFP also colocalized with the autophagic
vacule marker monodansylcadaverine (MDC), as
shown in (G) (LALP70/EGFP) and (H) (MDC).
Arrowheads in H indicate the transfected cells shown
in G. Cells were analysed 24 hours after transfection,
either by in vivo microscopy (A,B) or after fixation
(C-H). Analysis was performed either by confocal
microscopy (A-F) or by conventional fluorescence
microscopy (G,H). Bar, 10 µm (A,B,G,H); 5 µm (CF).
expression library and hUDPase from a brain expression
library, splice variants may be tissue-specific. Splice variants
were also proposed for a rat apyrase for which multiple bands
in northern blots from various tissues had been observed
(Kegel et al., 1997).
The LALP70 sequence as well as the hUDPase sequence
showed striking homologies to diphosphate phosphohydrolases
(E.C. 3.6.1.15), known as apyrases (Plesner, 1995). There are
five further human homolog proteins known: the cell surface
protein CD39 (21% homology; Maliszewski et al., 1994),
which has also been cloned from mice, three CD39-like genes
(CD39L1, 22% homology; Chadwick and Frischauf, 1997;
CD39L2, 20% homology and CD39L4, 22% homology;
Chadwick and Frischauf, 1998) and a brain ecto-apyrase
identical with CD39L3 (23% homology; Smith and Kirley,
1998). Other apyrases have been cloned from rat, chicken,
Caenorhabditis elegans, Toxoplasma gondii, yeast, potato and
pea, indicating that this protein family appeared early in
phylogeny and is spread over all kingdoms of eukaryotes.
Most apyrases are plasma membrane proteins with ectoNTP/NDPase activity. They are anchored in the membrane
with two transmembrane domains which are located at the N
and C terminus, respectively, constituting a type III membrane
protein (Singer, 1990). While two short amino acid sequences
point to the cytosol, the sequence between the transmembrane
domains faces the extracellular space. This extracellular part
of the protein contains four sequence stretches, highly
conserved between all known apyrases. They were described
as apyrase conserved regions (ACRs) by Handa and Guidotti
(1996). The LALP70 sequence also contains the four ACRs
(boxes in Fig. 3) as well as the N- and C-terminal
transmembrane domains. Computer analysis predicted two
possible N-terminal transmembrane sequences, one between
+3 and +25, and one between +37 and +53. In the in vitro
2482 A. Biederbick, S. Rose and H.-P. Elsässer
Fig. 7. Northern blot analysis using the Cterminal part of the LALP70 cDNA
(HindIII fragment from 1030 to 1702 bp)
for hybridization. (A) Dot blot matrix
representing RNA from 44 adult and 7
fetal human tissues which are indicated in
the table. Dots H1-H6 are negative
controls (H1, yeast total RNA; H2, yeast
tRNA; H3, E. coli rRNA; H4, E. coli
DNA; H5, poly r(A); H6, human C0t1
DNA). Intensities of the idividual dots
were quantitated with a phosphoimager
and are listed below (PLS). (B) Northern
blot of 15 µg of total RNA from PaTu
8902 cells separated on a 1%
formaldehyde gel, blotted on
nitrocellulose and hybridized with the
same cDNA as in A. Only one band was
obtained, representing a mRNA of about
7.4 kb.
# organ
A1 whole brain
A2 amygdala
A3 caudate nucleus
A4 cerebellum
A5 cerebral cortex
A6 frontal lobe
A7 hippocampus
A8 medulla
oblongata
B1 occipital lobe
B2 putamen
B3 substantia nigra
B4 temporal lobe
B5 thalamus
B6 nucleus
accumbeus
B7 spinal cord
C1 heart
C2 aorta
C3 skeletal muscle
translation experiment in the presence of microsomal
membranes no signal peptide was cleaved off, indicating that
both N-terminal transmembrane domains are maintained in the
mature protein. This is in accordance with reports about other
apyrases (Kegel et al., 1997). We assume that only the Cterminal end of the protein is facing the cytoplasmic site, while
there are two membrane-spanning domains at the N terminus
with the end of the protein pointing to the extracytosolic space.
Thus, LALP70 would be a type IIIb membrane protein (Singer,
1990; Fig. 8).
The molecular function of the ACRs are only partly
understood (Handa and Guidotti, 1996; Wang and Guidotti,
1998). The ACR1 and ACR4 domains are homologous to the
β- and γ-phosphate binding motif found in such different
proteins as actin, hsp70 and hexokinase (Flaherty et al., 1991).
Neither the ACRs nor other parts of the sequence are
homologous to the glycine-rich Walker consensus sequence for
ATP binding described for other ATPases (Walker et al., 1982),
indicating that the ACRs are critical for the enzymatic function
of apyrases. From Toxoplasma gondii an isoform was cloned
missing the ACR1 without alteration of the NTPase activity.
Moreover, an even more truncated apyrase was characterized
in pig pancreas containing only the ACR4 domain, but still
active as an apyrase (Kaczmarek et al., 1996). This indicates
that ACR4 harbors all capabilities necessary for a basal apyrase
function, while ACR1-3 might be involved in further
PLS
701
352
329
371
376
310
345
358
# organ
C4 colon
C5 bladder
C6 uterus
C7 prostate
C8 stomach
D1 testis
D2 ovary
D3 pancreas
PLS
441
168
280
504
748
1011
371
638
#
organ
PLS
E6 peripheral leukocyte
505
E7 lymph node
828
E8 bone marrow
242
F1 appendix
642
F2 lung
503
F3 trachea
658
F4 placenta
885
G1 fetal brain
431
967
472
556
376
431
522
D4
D5
D6
D7
D8
E1
pituitary gland
adrenal gland
thyroid gland
salivary gland
mammary gland
kidney
792
409
326
688
279
342
G2
G3
G4
G5
G6
G7
392
765
313
273
E2
E3
E4
E5
liver
small intestine
spleen
thymus
310
673
457
525
H4 E, coli DNA
H7 human DNA 100ng
H8 human DNA 500ng
fetal heart
fetal kidney
fetal liver
fetal spleen
fetal thymus
fetal lung
496
494
273
297
445
398
33
38
116
regulatory items of these enzymes. This was proposed by Wang
and Guidotti (1998), comparing amino acid sequence and
nucleotide substrate specificity of the ACR1 domain from
different apyrases. However, isoforms from Toxoplasma gondii
(NTP I and NTP II; Bermudes et al., 1994) differing in only
16 amino acids which are not located in the ACRs, differed
markedly in their ability to cleave ATP and ADP (75:1 in NTP
I versus 1:1 in NTP II). Although the LALP70 sequence differs
from the hUDPase sequence in only nine amino acids,
enzymatic activity of the LALP70 protein has still to be shown.
It will be of special interest whether the 9-amino-acid insert
influences the enzyme activity and/or substrate specificity.
Although apyrases were originally described as ectoenzymes being involved in the extracellular nucleotide
metabolism, the localisation at the plasma membrane has been
explicitly shown for only a few members of this family, i.e. for
CD39 (Kaczmarek et al., 1996), the rat and the human brain
apyrases (Kegel et al., 1997; Smith and Kirley, 1998).
However, six types of apyrases were associated with
intracellular compartments. The three apyrases NTP1-3 from
T. gondii and the apyrase from potato were located in
intracellular vacuoles, which have not been characterized in
detail. The apyrase from yeast (GDA1) and the hUDPase were
located in the Golgi apparatus, where they cleave UDP
molecules accumulating during protein glycosylation.
We used a cDNA for a LALP70/EGFP-fusion protein to
The human apyrase-like protein LALP70 is lysosomal 2483
N
R1
AC
2
R
AC R 3
4
AC
R
C
A
C
Fig. 8. Model of the insertion of LALP70 into the lysosomal
membrane as deduced from sequence alignment analysis,
hydrophobicity plot and in vitro transcription/translation
experiments. ACR1-4, apyrase-like regions in the N-terminal half of
the protein; green, C-terminal intraluminal part of the protein; blue,
transmembrane regions; red, 8 amino acid stretch unique for the
LALP70 protein. Arrows indicate possible trypsin cleavage sites in
the cytosolic domains. There are six possible cleavage sites which
are clustered near the transmembrane domains.
reveal the cellular localisation of this member of the apyrase
family in transient transfection experiments. The
LALP70/EGFP-fusion protein gave rise to an intracellular
punctate pattern resembling vacuolar structures. These
structures were concentrated in the perinuclear region in a
similar pattern to autophagic vacuoles stained with
monodansylcadaverine (MDC). When transfected cells were
counterstained with MDC, a partial colocalisation was
observed, indicating that the LALP70/EGFP-fusion protein is
located in a subset of the vacuoles of the lysosomal/autophagic
compartment. This was further confirmed in a colocalisation
study using an antibody against the lysosomal marker protein
lamp1 (Kornfeld and Mellmann, 1989). Using confocal
microscopy only a subpopulation of vacuoles were positive for
both the LALP70/EGFP-fusion protein and lamp1, while there
were also vacuoles either positive only for the LALP70/EGFPfusion protein or for lamp1. Localisation of the LALP70/
EGFP-fusion protein to the plasma membrane was not
observed under any conditions. Thus, LALP70 is located in a
subfraction of lysosomal/autophagic vacuoles. Since the
hUDPase described by Wang and Guidotti (1998) is almost
identical to LALP70, the structures that are LALP70/EGFPpositive and lamp1-negative might belong to the Golgi
apparatus. However, a localisation to the endoplasmatic
reticulum (ER) cannot be completely ruled out, especially
since the formation of autophagosomes originates most likely
from ER membranes (Dunn, 1990a). The difference between
the intracellular localisations of LALP70 and hUDPase might
depend on the nine different amino acids between these
proteins, which, as an alternative to their possible role for the
enzymatic activity (see above), could also act as a sorting
signals.
An intracellular apyrase function has only been shown for
the yeast apyrase GDA1 (Abeijon et al., 1993) and the
hUDPase (Wang and Guidotti, 1998). These Golgi proteins are
capable of cleaving GDP/UDP to GMP/UMP. GDP/UDP
accumulate from activated sugars used during protein
glycosylation. However, diphosphate nucleotides cannot cross
the Golgi membrane. In order to reutilize diphosphate
nucleotides they have to be converted to monophosphate
nucleotides, which then can be transported into the cytoplasm
across the Golgi membrane. Functional mutation of GDA1
results in accumulation of GDP in the Golgi cisternae and an
abrogation of protein glycosylation (Abeijon et al., 1993). The
lysosomal system receives material for degradation from
different sources like endocytosis, phagocytosis and
autophagocytosis. Especially during autophagocytosis, large
amounts of cytoplasm containing high concentrations of triand diphosphate nucleotides are sequestered. However, tri- and
diphosphate nucleotides cannot cross the lysosomal membrane
and have to be converted to nucleosides (Pisoni and Thoene,
1991). The lysosomal membrane protein acid phosphatase is
only capable of cleaving monophosphate nucleotides to
nucleosides, and cannot cleave higher phosphorylated
nucleotides (Pisoni, 1996). We propose that LALP70
participates in this part of nucleotide metabolism and is critical
for the salvage of nucleotides from the lysosomal/autophagic
vacuole lumen.
The lysosomal compartment is an endomembrane system
present in all eukaryotic cells. However, the total quantity of
this compartment and the ratio of subcompartments like late
endosome, phagolysosome or autophagolysosome differ
between cell types or in one cell type under different functional
conditions. The expression of LALP70 was analysed using a
dot blot northern hybridisation and also revealed a ubiquitous
distribution in 50 different human tissues, with only a sixfold
difference between the tissues with the highest and the lowest
expression. Similar results were obtained by Wang and
Guidotti (1998), analysing eight different human tissues. This
points to a general function presented in all cells, such as
turnover of nucleotides, and is compatible with our finding that
LALP70 is a lysosomal/autophagolysosomal membrane
protein.
We gratefully acknowledge the technical assistence of Uschi Lehr
and the preparation of the photographic reprints by Volkwin Kramer.
We thank R. MacDonald, H. F. Kern and T. Möröy for helpful and
critical discussions. This work was supported by Deutsche
Forschungsgemeinschaft, grant EL 125/1.
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