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J. Biol. Chem., Vol. 277, Issue 48, 46504-46511, November 29, 2002
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From the Biochemie-Zentrum Heidelberg,
Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld
328, D-69120 Heidelberg, Germany
Received for publication, July 12, 2002, and in revised form, September 9, 2002
The p24 proteins belong to a highly conserved
family of membrane proteins that cycle in the early secretory pathway.
They bind to the coat proteins of COPI and COPII vesicles, and are proposed to be involved in vesicle biogenesis, cargo uptake, and quality control, but their precise function is still under debate. Most
p24 proteins form hetero-oligomers, essential for their correct localization and stability. Functional insights regarding the mechanisms of their steady state localization and the role of interaction with coat proteins has been hampered by a lack of data on
their concentration and state of oligomerization within the
endoplasmic reticulum, the intermediate compartment, and Golgi complex. We have determined for all mammalian p24 family
members the size of the oligomers formed and their stoichiometric
relation in each of these individual organelles. In contrast to earlier reports, we show that individual members exist as dimers and monomers and that the ratio between these two forms depends on both the organelle investigated and the p24 protein. We find unequal quantities, with p23 and p27 building up concentration gradients, ruling out a
simple 1:1 stoichiometry. In addition, we show differential cycling of
individual p24 members. These data point to a complex and dynamic
system of altering dimerizations of the family members.
The p24 family of type I transmembrane proteins consists of six
members in mammalian cells which, referring to sequence homologies, can
be divided in four subfamilies (1, 2). Mammalian cells contain three
members of the p26 subfamily, namely p26, p27, and tp24 and only one
member each of the p23, p24, and p25 subfamilies. According to studies
in yeast, p24 proteins may function as cargo receptors or adaptors
since in knock-out mutants distinct cargo proteins show delayed
transport kinetics (3-5). Additional experiments in yeast and
Caenorhabditis elegans implicate also an involvement of p24
proteins in ER1 quality
control (4, 6, 7). For mammalian p23, it has been proposed that it
serves as COPI receptor because it can bind to coat components (1, 8)
and belongs to the minimal machinery needed to bud COPI vesicles from
liposomes (9). In addition, a direct interaction of p24 family members
has been shown with proteins involved in vesicle budding and cargo
sorting, such as ARF-1 (ADP-ribosylation factor) (10, 11), ARF-GAP
(GTPase-activating protein) (10) and Sar-1p (12). p24 proteins are also
effectively enriched in COPI (8, 13) and COPII vesicles (3, 5), and
some of them bear signals in their cytoplasmic tails, which when fused
to a cargo protein direct them either anterogradely or retrogradely
(14). Therefore, p24 proteins have been implicated to be involved in
transport processes of the early secretory pathway, but their precise
function is still unclear.
All p24 proteins are found in membranes of the early secretory pathway
(1, 8, 13, 15, 16), and there is evidence that they cycle
constitutively between these membranes (13, 14, 16, 17). Another
outstanding property of p24 proteins is the formation of
hetero-oligomers. As was shown in several immunoprecipitation studies,
they interact with each other to a certain extent (3, 4, 13, 16).
Moreover, overexpression of a single p24 protein leads to a
mislocalization of all p24 proteins in ER-derived structures (13, 15,
16), and only the simultaneous overexpression of p24 proteins of all
subfamilies results in a convincing perinuclear Golgi localization (15, 16). Furthermore, in yeast cells lacking one p24 protein (4) and in
cells from mice lacking one allele of p23 (18) other family members are
degraded. Thus, there is a strict dependence among p24 proteins in
terms of stability, transport, and/or localization. Consequently, the
formation of hetero-oligomers seems to be a prerequisite for their
correct function.
To set a basis for understanding the molecular functionality of p24
proteins, we have undertaken a detailed study of their oligomeric
behavior, their stoichiometric relation, and cycling in the membranes
of the secretory pathway. We demonstrate that p24 proteins, in contrast
to earlier results (1, 4), exist either as dimers or monomers with no
higher oligomers observed. Moreover, we find different ratios between
dimers and monomers depending both on a given p24 protein and on its
subcellular localization. p23 and p27 build up concentration gradients,
and p24 and p25 are distributed equally in the early secretory pathway,
with differential cycling between these compartments. Therefore, we
assume highly dynamic and complex interactions of these four p24
proteins. In contrast, p26 and tp24 do not seem to interact with the
other four members as they occur exclusively either as monomer or as dimer, respectively, and their concentrations are vastly different from
other p24 proteins.
Antibodies--
Antibodies directed against p23, p24, p25, p26,
tp24, and p27 were raised in rabbits, chicken, and guinea pigs with the
peptides or recombinant proteins indicated in parentheses
coupled to keyhole limpet hemocyanin (Sigma): Henriette (p23
recombinant luminal domain), HAC344 (p23-tail, CLRRFFKAKKLIE), Thelma
(p23, KITDSAGHILYSK), #1327chicken (p23, KITDSAGHILYSK), Elfriede (p24
tail, (13)), Frieda (p24 tail, CYLKRFFEVRRVV), #1593chicken (p24,
CYLKRFFEVRRVV), #2469R1 (p25-tail, CYLKSFFEAKKLV), #2088M2 (p26-tail,
CLLKSFFTEKRPISRHVHS), #2087R2 (p27-tail, CLLKSFFSDKRTTTTRVGS), #2501R2
(tp24 tail, CTLKRFFQDKRPVPT). Immunization and affinity purification
was performed according to standard protocols (19). Rabbit polyclonal
antibodies against cytochrome b5 were a kind gift of N. Borgese
(University of Milan, Italy), monoclonal antibodies against ERGIC-53
were from H.-P. Hauri (Biocenter, Basel, Switzerland) (20), and rabbit
polyclonal antibodies against p30 (mitochondrial protein) and PMP69
(peroxisomal membrane protein) were provided by W. Just
(Biochemie-Zentrum Heidelberg, University of Heidelberg, Germany).
Further antibodies used were: anti-protein disulfide isomerase (PDI, BD
Transduction Laboratories), anti-KDEL-receptor (Stressgen
Biotechnologies), anti-transferrin receptor (Zymed
Laboratories Inc.), anti-rabbit-peroxidase (POD, BioRad
Laboratories), anti-mouse-POD (Dianova), anti-guinea pig-POD (Dianova),
anti-chicken-POD (Dianova), monoclonal anti-rabbit-POD (Sigma),
anti-mouse-Alexa-488 (Molecular Probes), and anti-guinea pig-Alexa-456
(Molecular Probes).
Subcellular Fractionation--
HeLa cells (ATTC: CCL-2.2) were
grown in Spinner culture according to standard conditions up to
4-6 × 105 cells/ml. Homogenization and
centrifugation were performed according to Ref. 16. After
centrifugation, 18 instead of 9 fractions were taken from the top of
the gradient and were diluted 1:1 with buffer and centrifuged for
1 h at 100,000 × g at 4 °C. Membrane pellets
were used for further analysis.
Galactosyltransferase activity was measured according to Ref. 21, and
alkaline phosphodiesterase corresponding to Ref. 22. The content of
other marker proteins was determined by Western blot analysis with the
appropriate antibodies using the ECL detection system (Amersham
Biosciences). The signals were quantified with the
QuantityOne® software from BioRad Laboratories, and the relative protein amounts were calculated.
Solubilization and Gel Filtration--
Solubilization conditions
were tested with 50 µg of rabbit liver Golgi membranes (23). After
pelleting, the membranes were solubilized in 50 µl of the indicated
buffers (containing 3% 8-n-octyl-poly-oxyethylene (POE), Alexis
Corporation; 2% cholic acid, Sigma; or 4% octylglucoside (n-octyl-
Membrane pellets (equivalent to 50 nmol of phospholipid as determined
by phosphate measurement) (24) from subcellular fractionations were
solubilized for 30 min on ice in 4% octylglucoside, 20 mM Hepes, pH 6.8, 50 mM NaCl, unless indicated differently.
After centrifugation (1 h, 100,000 × g at 4 °C),
the supernatant was fractionated on a Superdex 200 column on the SMART
system (Amersham Biosciences) using a flow rate of 50 µl/min and 1%
octylglucoside, 20 mM Hepes, pH 6.8, 50 mM NaCl
as running buffer. All fractions were trichloroacetic
acid-precipitated, applied to SDS-PAGE (25), and further analyzed by
Western blotting with several p24 protein antibodies (Henriette,
Elfriede, #2469R1, #2088M, #2087R2, #2501R2). The column was
calibrated with gel filtration standards from BioRad Laboratories
(thyroglobin (670 kDa), bovine- Chemical Cross-link with Disuccinimidyl Glutarate (DSG)--
ER,
intermediate compartment (IC), and Golgi membranes according to a
phospholipid content of 6.25 nmol were diluted in phosphate-buffered saline and pelleted for 1 h at 100,000 × g at
4 °C. Pellets were resuspended in phosphate-buffered saline and
treated either with DSG (final concentration 0.6 mM) in
Me2SO or Me2SO alone for 30 min on ice.
Tris-HCl, pH7.5, was added to stop the reaction, and the samples were
further analyzed by Western blotting using an anti-p23 antibody (Thelma).
Immunoprecipitation--
Immunoprecipitations were carried out
according to Ref. 13. In contrast to this study, the antibodies
were covalently coupled to the protein A-Sepharose (19).
GST-p24 Tail Constructs and Luminal Domain of p23--
Sense and
antisense oligonucleotides were synthesized corresponding to the
cytoplasmic tail sequences of p24, p25, p26, p27, and tp24 with
overhanging restriction sites for EcoRI and
BamHI. After annealing of the oligonucleotides they were
cloned into the EcoRI and BamHI site of the
pGEX-2T vector (Amersham Biosciences). The GST-p24 tail constructs were
overexpressed and purified according to manufacturers' specifications.
The sequence for the luminal domain of p23 was cloned into the
BamHI and HindIII site of the pQE-30 vector
(Qiagen), overexpressed, and purified by nickel-nitrilotriacetic acid-agarose. The purity of the overexpressed proteins was tested by
Coomassie staining, and protein concentration was determined by the
Bradford method (BioRad Laboratories).
15 °C Block and Release at 37 °C--
For
immunofluorescence studies, Vero cells (ATTC CCL-81) were cultured on
coverslips, incubated for 2 h at 15 °C in carbonate-free Hepes-buffered standard medium and afterward transferred to 37 °C
for various time periods. Cells were then fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100, and labeled with p24 protein antibodies. They were affinity purified and anti-p23 (HAC344), -p24
(Elfriede), and -p25 (#2469R1) directly labeled with cy3, anti-p27
(#2087R2) with cy5 according to the manufacturers specifications (Amersham Biosciences). Additional antibodies used were detected with
Alexa-488/456-coupled secondary antibodies. For confocal analysis a
Leica TCS-SP with the Leica confocal software was used.
For Western blot analysis s-HeLa cells were grown in Spinner culture,
transferred in 15 °C cold, carbonate-free, Hepes-buffered standard
medium, and incubated at 15 °C for 2.5 h. Cells were harvested
at various time points, and subcellular fractionation was performed as
described before. For each condition the distribution of marker enzymes
was tested, and fractions were pooled accordingly. The amount of
membrane in each pool was determined by nano-ESI-MS/MS (electrospray ionization-tandem mass spectrometry) (27), and identical
amounts based on phosphatidylcholine content were subjected to Western
blot analysis with p24 protein antibodies (Henriette, Elfriede,
#2469R1, #2088M2, #2087R2, #2501R2), using the ECL detection system.
The signals obtained were quantified with QuantityOne software, and the
signals for each compartment were used to calculate relative protein amounts.
Quantitative Western Blot Analysis--
GST-p24 tail constructs
(p24, p25, p26, p27, and tp24) or the luminal domain of p23 were used
as p24 standard proteins. Increasing amounts of standard protein (in
ng) and membranes (in nmol of phospholipid) were subjected to gel
electrophoresis (28) and Western blotting with antibodies against p24
proteins (Henriette, Frieda, #2469R1, #2088M, #2087R2, #2501R2), and
developed with the ECL detection system. The antibodies had been tested
before to be specific for each p24 protein and not to cross-react with GST. The signals were quantified with the QuantityOne software, and
standard curves with the data of the p24 standards were established. Only values of the membrane samples within these linear curves were
taken into account to calculate the concentration of each p24 protein
in ng of protein/nmol of phospholipid (see supplemental data Figure I
at http://www.jbc.org). For each protein, at least three
independent estimations were made, each time with both the standards
and the samples on the same blot. Independent experiments with
different p24 protein antibodies in a second quantitative Western blot
analysis were performed as an additional control, and comparable
results were obtained (supplemental data Figure II).
Subcellular Fractionation of ER, IC, and Golgi--
To fractionate
ER, IC, and Golgi, HeLa cell membranes were subjected to density
gradient centrifugation. As shown in Fig. 1, these membranes of the early secretory
pathway are well separated. Golgi membranes were found in light
fractions (fractions 3 + 4), characterized by galactosyltransferase
activity and the KDEL receptor (KDEL-R.), and are separated
from plasma membrane in fraction 2 as shown by alkaline
phosphodiesterase (AP) activity. The second peak of the KDEL
receptor in the intermediate density fractions represents the membranes
of the IC, as this protein localizes to the cis-Golgi and the IC,
respectively (29, 30). In contrast, ER membranes move to the high
density region of the gradient (fractions 15-17), as shown by the
distribution of PDI. The ER pool also contains mitochondria and
peroxisomes as indicated by the distribution of p30 and PMP69. However,
the amount of membrane according to peroxisomes in ER fractions can be
disregarded as these membranes only constitute 1% of total membranes
(31). In contrast, mitochondria represent about 32% of total cell
membranes (estimated for baby hamster kidney cells, (31)) and thus only
part of the membranes in the ER pool represents ER membranes. This was
quantified and taken into account.
Solubilization and Oligomeric Behavior of p24 Proteins in ER, IC,
and Golgi--
Three different detergents were tested under the same
buffer and salt conditions described before for the solubilization of p24 proteins (1, 4, 32). As shown in Fig.
2, only octylglucoside and 8POE were able
to solubilize p23 almost quantitatively. Cholic acid, used
previously to determine an oligomeric size of mammalian p24 proteins of
35S (1) in our hands emerged to be unsuited for solubilization. Since
an extensive study of p24 proteins in yeast was performed with
octylglucoside, and this detergent solubilizes p23 nearly
quantitatively (Fig. 2) (4), it was used for solubilization in further
experiments. Equal amounts of ER, IC, and Golgi with regard to their
phospholipid content were solubilized in 4% octylglucoside, and the
p24 oligomers were separated by gel filtration. The fractions were
subsequently analyzed by Western blotting with antibodies against all
p24 proteins. As shown in Fig. 3, p24
proteins elute at two defined volumes with peaks in fractions 18 and
21. To determine the size of the p24 oligomers in these two peaks, we
calibrated the gel filtration column with membrane proteins rather than
soluble proteins, which, like p24 proteins, need a detergent micelle to stay in solution (Fig. 4A).
With reference to this calibration, p24 proteins appear as dimers and
monomers in the ER, IC, and Golgi pools. To assess the oligomeric state
of p23 independently, i.e. without the use of detergent, (i)
soluble recombinant luminal domain of p23 was analyzed by gel
filtration in the absence of detergent and (ii) ER, IC, and Golgi
membranes were treated with a cross-linker (DSG) and afterward analyzed
by Western blotting with an anti-p23 antibody (supplemental data Figs.
III and IV). In both detergent-independent experiments a dimer is the
highest oligomer observed. This clearly shows that the dimer observed in the gel filtration analysis is physiological and is not a result of
the disruption of higher oligomers by the use of detergent.
In addition, we observed a different ratio between dimer and monomer,
depending on the organelle investigated. As depicted in Fig.
3B, p23 is predominantly found as a dimer in the ER, but in
the IC and the Golgi there are about equal amounts of dimer and
monomer. In contrast, p24 shows a similar ratio between monomer and
dimer in all membrane fractions. p25 and p27 exist predominantly as
monomers, with p25 showing the highest amount of dimer in the ER and
p27 in the Golgi. However, p26 and tp24, which coimmunoprecipitate to a
significantly lower extent with other p24 members (16), are
present only as either monomers or dimers, respectively.
As shown in Fig. 5 an anti-p24 antibody
coimmunoprecipitates p23 and p25, and an anti-p27 antibody p23 and p24
from all compartments of the early secretory pathway. Therefore, a part
of p24 proteins always exists as heterodimers, but the amount of the
coimmunoprecipitated p24 protein is different depending on the
organelle. For example, when compared with the Golgi, p27
coimmunoprecipitates less p24 than p23 from the IC. This implies that
in the different organelles certain heterodimers are preferentially
formed.
Cycling of p24 Proteins in the Early Secretory Pathway--
All
members of the p24 family cycle in the early secretory pathway (13, 14,
16, 17) and accumulate in the IC after incubation of cells at 15 °C
(13, 16, 17). After release of this 15 °C block by shifting to
37 °C, p24 proteins start to move to their steady state
localization. This system was used as a tool to compare the cycling of
all p24 proteins by immunofluorescence and Western blot analysis. For
immunofluorescence studies, cells were fixed after a 15 °C block for
2 h and after various time periods of release at 37 °C. Then
the cells were triple-labeled with antibodies against p27, ERGIC-53,
and a second p24 member and analyzed by confocal microscopy. As shown
in Fig. 6A, at
15 °C p24 and p27 colocalize with the ERGIC-marker ERGIC-53 in a perinuclear region (yellow arrowhead) and peripheral
punctate structures (yellow arrows). After shifting the
cells to 37 °C, the colocalization with ERGIC-53 in the perinuclear
region and later in punctate structures decreases, as parts of p24 and
p27 are redistributed to the Golgi (white arrowheads, Fig.
6A, I and II). In contrast, p23 and p25 (Fig. 6A,
III and IV, for p25 supplemental data Fig. IV) at 15 °C are
localized to the perinuclear region where they colocalize with p27 and
ERGIC-53, but they are also found in reticular structures likely of the
ER (blue arrowheads). After shifting to 37 °C, parts of
p23 and p25, such as p24 and p27, are transported to the Golgi with
some of p23 and p25 still found in reticular structures. This clearly
shows that p24 proteins travel differentially in the early secretory
pathway, with p24 and p27 mainly cycling between the IC and the Golgi
and p23 and p25 accumulating in the IC and reticular structures of the
ER, indicating that they are either transported retrogradely or that their appropriate partner for further transport (for example p24) is
blocked elsewhere. p26 and tp24 behave similar to p24 and p27, but show
a pronounced punctate staining pattern (data not shown).
To analyze the cycling of p24 proteins biochemically, subcellular
fractionation was performed after a temperature block at 15 °C and
at several time points after transferring the cells to 37 °C. As
described under "Experimental Procedures," the relative amounts of
p24 proteins were compared by Western blot analysis (Fig.
6B). In agreement with the immunofluorescence studies, p23 and p25 are both constitutively exported from the ER (Fig.
6B). On the other hand, the amount in the ER of p27
increases with the release, and p24 shows no significant change of
relative protein amounts in the ER during the time course. In the IC,
p25 redistributes like p24 and p27, as all three members seem to be
transported into this organelle. However, the amount of p23 first
increases and then decreases in this compartment. In Golgi membranes,
the relative amounts of p25, p24, and p27 decrease after a 5-min
release and then increase after 15 min but, in every case, to a
markedly different extent. In contrast, the relative amount of p23
decreases steadily. This demonstrates that p24 proteins cycle
differentially, but at some transport steps also behave similarly, such
as p23 and p25 in the ER.
Localization of p24 Proteins and Their Concentration in the
Individual Organelles of the Early Secretory Pathway--
Several
independent studies show that all p24 proteins localize to the various
membranes of the early secretory pathway (1, 8, 16, 17, 33), but it is
still not clear to what extent p24 proteins reside within the same
compartment. Therefore, we addressed both their localization and
concentration by quantitative Western blot analysis. To this end, ER,
IC, and Golgi were enriched by subcellular fractionation of HeLa cells
as described above (Fig. 1). As exemplified with p27 (see supplemental
data Fig. I), defined amounts of membrane according to phospholipid
content and p24 protein standards were subjected to Western blot
analysis with their respective antibodies. After quantification of the signals, a standard curve with the data of the p24 protein standards was obtained, and only values of the membrane samples within the linear
range of this curve were used to calculate the concentration of a p24
protein. In Fig. 7, the average
concentrations of all p24 proteins in ER, IC, and Golgi are summarized.
Two p24 members, namely p25 and p24, are equally distributed between
ER, IC, and Golgi, whereas p23 and p27 built up opposing gradients
across the early secretory pathway. In addition, p23 and p25, the only members with functional ER retrieval signals in their cytoplasmic tails
(14, 34, 35), exist in a 5-fold lower concentration than p24 and p27.
However, p27 is present in the ER at the same concentration as p23 and
p25, due to its concentration gradient along the early secretory
pathway. In contrast, p26 exists in very high and tp24 in very low
concentrations in all three organelles, again pointing to a less
related function of these p24 proteins.
p24 proteins present a highly conserved protein family of small
type I transmembrane proteins, all with small cytoplasmic domains.
Although it is in general accepted that they play a role in transport
processes of the early secretory pathway, their precise function is
still unclear. Prominent properties of p24 proteins are their
constitutive cycling in the early secretory pathway (14, 16, 17, 33)
and the capability to form hetero-oligomers with each other (3, 4, 13,
16). Studies in yeast and in mammals show that in vivo the
amount of one p24 protein is regulated depending on other p24 proteins
(4, 18). In addition, overexpression of a given p24 protein results in
a mislocalization of other p24 proteins in ER-derived structures, and
only the overexpression of at least one member of each subfamily leads
to their clear-cut localization to the Golgi apparatus (15, 16). Thus,
most of the p24 proteins depend on each other in terms of stability,
localization, and/or transport. Hence, the formation of
hetero-oligomers seems to be intimately coupled to their function.
p26 and tp24 Seem Not to Require Hetero-oligomers for Their
Function--
In contrast to other p24 proteins, p26 and tp24 do not
seem to require the formation of hetero-oligomers for their
function, as they do not significantly coimmunoprecipitate (16),
and are not mislocalized when other p24 proteins are overexpressed
(32). We demonstrate here that also their oligomeric behavior and
stoichiometric relation, compared with other p24 proteins, are pointing
to a different molecular functionality. p26, which occurs mainly as a
monomer and in much higher concentrations than all other members, is
distributed more or less equally among all membranes. Therefore, it
might serve as a placeholder in budding zones building up a sorting
and/or budding competent matrix by interacting with other p24 proteins
only transiently. tp24 is set apart from other members by its
occurrence only in miniscule concentrations.
Oligomeric State of p24 Proteins in ER, IC, and Golgi
Membranes--
We investigated the oligomeric behavior of all
mammalian p24 proteins in the membranes of the early secretory pathway
by gel filtration analysis. In an earlier study, a hetero-oligomer of p24 proteins with a sedimentation coefficient of 35S was determined in
mammalian cells, which would correspond to a complex of around fifty
p24 proteins (1). However, in our hands the detergent used in this
study appeared to be unsuited for solubilization of p24 proteins, as
after centrifugation at 100,000 × g for 30 min most of
p23 remain insoluble. Therefore, the determination of a 35S complex is
very likely caused by partly solubilized membranes. In further studies,
a size of about 100 kDa for a p24 oligomer in yeast and p23 in
mammalian cells was determined (4, 32), and therefore it was proposed
that p24 proteins form tetramers. In contrast to these earlier studies,
we demonstrate that p24 proteins form dimers and monomers in the
membrane of ER, IC, and Golgi (Fig. 3). The differing results are due
to the fact that we have calibrated the gel filtration with membrane
proteins rather than with soluble proteins. Calibration with soluble
proteins, as shown in Fig. 4B, leads to an overestimation of
the oligomer size caused by an increase of the apparent molecular mass
by the detergent micelle in the membrane protein samples. To
challenge our results, the soluble luminal domain of p23 was also
subjected to a gel filtration analysis calibrated with soluble standard proteins in the absence of detergent (supplemental data, Fig. III), and
mainly a dimer was found. Moreover, in a cross-link experiment
performed with p23, a band of around 45 kDa was observed, which
corresponds to a dimer and confirms our results. Altogether this is
taken as detergent-independent evidence that a p24 oligomer indeed
is a dimer and not the result of detergent-induced disruption of higher oligomers.
Several independent studies show that p24 proteins can be
coimmunoprecipitated with each other (3, 4, 13, 16). As p27 can
coimmunoprecipitate p23, p24, and p25, it was concluded that these p24
proteins form a 1:1:1:1 tetramer (16). However, our data imply that
there are heterodimers of p23, p24, p25, and p27 rather than the
formerly proposed tetramer (4, 16), which is also in line with the
coimmunoprecipitation data. In vitro studies show that p24
protein cytoplasmic tails can form tetramers (36), which seems to be
functionally important as a tetramer of p23 cytoplasmic tails is able
to induce a conformational change in coatomer (37). On the other hand,
it has been reported that in vivo an interaction of p24
proteins is mediated by the coiled-coil domains in the luminal part of
p24 proteins (15), and not by their cytoplasmic tails. Therefore, we
conclude that tetrameric states of p24 proteins mediated by the
cytoplasmic tails are of transient nature, and are obviously not stable
in the detergent conditions used in the gel filtration analysis.
For p23, p24, p25, and p27, a different ratio of dimer to monomer was
observed depending on the p24 protein and the organelle investigated
(Fig. 3). In addition, the question arises of whether p24 proteins
exist as homodimers or heterodimers. In Fig. 5 we show that
heterodimers exist in all compartments of the early secretory pathway,
whereas the ratio of coimmunoprecipitated p24 proteins is different
depending on the organelle. This implies an environment-specific
binding preference of certain p24 proteins.
There is evidence from independent studies that p24 proteins might
function as dimers as only the dimeric form of a peptide resembling the
cytoplasmic tail of p23 inhibits the recruitment of ARF1-GDP to
Golgi membranes (10) and only a dimer of p24 binds to Golgi matrix
proteins (38). Therefore, it could be presumed that the dimeric form of
these family members is a functional one. This then would signify that
some of them, like p24, function at all levels of the secretory pathway
as they always show the same ratio between monomeric and dimeric
state. Others like p23, which exists mainly as a dimer in the ER and
displays an equal ratio between monomer and dimer in the other
compartments, would then play a role only at certain stages. This is
also in line with a concentration gradient of p23 between ER and Golgi
and the equal distribution of p24 to all compartments. As discussed above, by interaction of cytoplasmic domains of some p24 proteins transient formation of tetramers might occur.
Stoichiometry of p24 Proteins in the Organelles of the Early
Secretory Pathway--
As p24 proteins depend on each other in terms
of stability (4, 18), localization, and/or transport (15, 16), a
stoichiometric relationship of these proteins has always been presumed.
To investigate this relationship, the concentration of all p24 proteins
in the membranes of the early secretory pathway was determined by
quantitative Western blot analysis (Fig. 7). We demonstrate that p24
proteins are present in unequal concentrations and localize to
differing extents to membranes of ER, IC, and Golgi (Fig. 7), which
excludes a simple 1:1 stoichiometry. p23 and p25, both containing
functional ER retrieval signals in their cytoplasmic tails (14, 34,
35), exist in 5-fold lower concentration in the membranes of the early secretory pathway than the "anterograde" directed proteins p24 and
p27 (1, 14). p24 and p25 localize equally to all membranes; p23
and p27 build up opposing gradients with p23 present in higher concentrations in the ER and IC than in the Golgi and p27 with higher
amounts in the Golgi and the IC than in the ER. In the ER p27 exists in
mass amounts equal to p23 and p25. In this regard the family members
resemble the SNARE proteins, which also build up gradients (39). As p23
contains a functional retrieval signal in its cytoplasmic tail (14), it
is probably active in the "retrograde" transport processes from IC
to ER, and p27 as an anterograde p24 protein from the IC to the
Golgi. In contrast, the equally distributed p24 and p25 seem to have a
function in all organelles of the early secretory pathway. p25,
carrying a classical ER retrieval signal may prevent other
anterogradely directed p24 proteins from being transported to as far as
the plasma membrane, and thus ascertain their localization to the
membranes of the early secretory pathway. Then p24, with no functional
retrieval signal (14), may serve in the opposite direction and
facilitate the transport of retrograde members in the anterograde
direction. To simplify the quantitative aspect of our data, the
relative mass amounts found for p23, p24, p25, and p27 within the
individual organelles of the early secretory pathway are
depicted schematically in Fig.
8.
Cycling of p24 Proteins within ER, IC, and Golgi
Membranes--
The redistribution of p24 proteins after a 15 °C
block and subsequent release at 37 °C implies that p24 proteins
cycle differentially in the early secretory pathway but at certain
transport steps seem to behave similarly. For example, p23 and p25 upon
temperature block are distributed more retrogradely to ER membranes
(Fig. 6A) and are transported out of the ER to the same
extent after release of the block (Fig. 6B). On the other
hand, they show unequal redistribution to the IC and the Golgi. Hence,
it is impossible that they continuously form p23/p25 heterodimers. On
the other hand, p25 is redistributed like p24 and p27 to the IC and
Golgi and may then form hetero-oligomers with these p24 members. This altogether implies highly dynamic interactions of various family members.
Molecular Functionality of p23, p24, p25, and p27--
p23, p24,
p25, and p27 need each other for their correct localization and
stability (4, 15, 18). Taking into account (i) their formation of
heterodimers, (ii) differential cycling, and (iii) varying ratios of
dimers and monomers in the membranes of the early secretory pathway,
one explanation for this behavior would be a frequent alteration of
binding partners, likely to depend on the transport step, and resulting
in various dimeric combinations (heterodimers AB, AC, ... and
also possibly homodimers). Some of the p24 proteins contain functional
retrieval signals in their cytoplasmic domains (14, 34, 35), and they
are effectively incorporated into COPI (8, 13) and COPII vesicles (3).
Therefore, it is tempting to speculate that the composition of a p24
dimer rules the direction of a vesicle. p23 and p27, which build up
opposing gradients between ER, IC, and Golgi, could fulfill this
function, and p24 and p25, which are equally distributed, would serve
as anchoring partners to keep them in place until they are incorporated
into vesicles or mediate their transport back to their place of function.
We thank N. Borgese, W. Just, and H.-P. Hauri
for kindly providing antibodies, C. Reinhard and W. Nickel for helpful
discussions and critically reading the manuscript, and R. Pepperkok
and D. Lay for help with the confocal microscopy.
*
Work in the laboratory of the authors is supported by grants
of the German Research Foundation (to B. B. and F. T. W.), the Human
Frontiers Science Program Organization (to F. T. W.), and the Peter
and Traudl Engelhorn Foundation (to B. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 16, 2002, DOI 10.1074/jbc.M206989200
The abbreviations used are:
ER, endoplasmic
reticulum;
PDI, protein disulfide isomerase;
POD, peroxidase;
DSG, disuccinimidyl glutarate;
IC, intermediate compartment;
GST, glutathione S-transferase;
POE, n-octyl-poly-oxyethylene;
SNARE, soluble NSF attachment protein receptors.
Oligomeric State and Stoichiometry of p24 Proteins in the
Early Secretory Pathway*,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside), Calbiochem), incubated for
30 min on ice and subjected to 100,000 × g
centrifugation for 30 min. Pellets and the trichloroacetic
acid-precipitated supernatants were then analyzed by Western blotting
with an antibody against p23 (Henriette).
-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1.35 kDa)) for calibration with membrane proteins. Golgi membranes from rabbit liver (23) and from Chinese hamster ovary cells containing a CD8-Myc-construct (CD8-LT) (26), were mixed, pelleted,
solubilized, and analyzed by gel filtration as described before.
Thereafter, the fractions were subjected to Western blot analysis with
antibodies against transferrin receptor (190 kDa), Calnexin (90 kDa),
Myc (CD8-LT, 60 kDa) and cytochrome b5 (15 kDa).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
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Fig. 1.
Subcellular fractionation of ER, IC,
and Golgi membranes. HeLa cell membranes were subjected to density
centrifugation on a Nycodenz gradient. For each fraction
galactosyltransferase (Gal-T., cross) and
alkaline phosphodiesterase (AP, filled square)
activity were tested. The amount of KDEL-receptor
(KDEL-R., open rhombus), PDI (filled
triangle), PMP69, (open square), and p30 (open
circle) were determined by Western blot analysis of each fraction
with the appropriate antibodies. Golgi membranes peak in fractions 3 and 4, IC membranes in fractions 8-10, and ER membranes at high
density in fractions 15-17. In the high density fractions,
mitochondria (p30) and peroxisomes (PMP69) are also found.
View larger version (26K):
[in a new window]
Fig. 2.
Solubilization of p23. Rabbit liver
Golgi membranes were subjected to various buffer and detergent
conditions as described under "Experimental Procedures" and
centrifuged, and supernatants and pellets were analyzed by Western
blotting with antibodies against p23.
View larger version (34K):
[in a new window]
Fig. 3.
Gel filtration analysis of p24 proteins.
A, 50 nmol of ER, IC, and Golgi membrane according to
phospholipid content were solubilized in 4% octylglucoside and
separated on a Superdex 200 column. All fractions were trichloroacetic
acid-precipitated and applied to Western blot analysis with p24
antibodies. B, D and M illustrate
ratios of dimer to monomer. Bold letters indicate the
prevailing form.
View larger version (16K):
[in a new window]
Fig. 4.
Calibration of the gel filtration
column. The gel filtration column was either calibrated using
membrane proteins (A) or soluble proteins (B), as
indicated under "Experimental Procedures." Note that the presence
of detergent leads to an overestimation of the size of membrane
proteins.
View larger version (53K):
[in a new window]
Fig. 5.
Coimmunoprecipitation of p24 proteins from
ER, IC, and Golgi. Anti-p24 (A) and anti-p27
(B) were used to coimmunoprecipitate other p24 proteins from
ER, IC, and Golgi membranes. The precipitated material was analyzed by
Western blotting with the indicated antibodies.
View larger version (37K):
[in a new window]
Fig. 6.
Analysis of p24 protein cycling in the
early secretory pathway. A, immunofluorescence analysis.
Vero cells cultivated on coverslips were fixed after 2 h of
incubation at 15 °C or 15 min release at 37 °C after 15 °C
block. Then cells were triple-labeled with antibodies against ERGIC-53
(green), p27-cy5 (blue), and p24 cy3 or p23-cy3
(red). The distribution of p24 (I + II) and p23 (III + IV)
and simultaneously ERGIC-53 (green) and p27-cy5 (blue) are shown.
Yellow arrowheads mark the perinuclear structures, and
yellow arrows the punctate structures of the IC
characterized by ERGIC-53. Blue arrowheads highlight
reticular structures likely of the ER, and white arrowheads
point to structures that belong to the Golgi and do not overlap exactly
with the ERGIC-53 labeling. B, Western blot analysis. s-HeLa
cells were grown in Spinner culture and incubated for 2.5 h at
15 °C or 2.5 h at 15 °C plus 5 min or 15 min at 37 °C.
Then the cells were subjected to subcellular fractionation as described
under "Experimental Procedures," and ER, IC, and Golgi fractions
were pooled. Identical amounts of membranes according to their
phosphatidylcholine content were analyzed by Western blotting with p24
antibodies. The signals were quantified, and for each organelle the
values of the individual time points were used to calculate relative
protein amounts.
View larger version (41K):
[in a new window]
Fig. 7.
Concentration of p24 proteins.
Concentrations of p24 proteins in the ER, IC, and Golgi are summarized.
For each membrane and p24 protein at last three independent estimations
were made, each time with both the standard and the membrane samples on
the same blot. In the diagram mean values of all experiments and the
standard deviation as error bars are shown. In the table
below the values are summarized.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
[in a new window]
Fig. 8.
Schematic depiction of the
relative molar amounts of p23, p24, p25, and p27
within the ER, IC, and Golgi apparatus of a mammalian cell.
ACKNOWLEDGEMENTS
FOOTNOTES
The on-line version of this article (available at
http://www.jbc.org) contains Figures I-V and their legends.
To whom correspondence should be addressed. Tel.: 0049-6221544160;
Fax: 0049-6221544366; E-mail:
Felix.Wieland@urz.uni-heidelberg.de.
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