(Received for publication, August 4, 1995; and in revised form, January 9, 1996)
From the
The coat protomer complex I (COPI) family of coat proteins are
involved in the assembly of membrane-associated coats thought to
mediate vesicular transport between the endoplasmic reticulum and the
Golgi complex, between adjacent Golgi cisternae, and possibly in the
endocytic pathway. We investigated whether this heterogeneity in the
sites of COPI action might be reflected in biochemical heterogeneity of
one or more COPI subunits. A simplified method was devised to purify
the cytosolic COPI precursor complex, coatomer, from rat liver cytosol.
The individual subunits were analyzed by high resolution two
dimensional gel electrophoresis and mass spectroscopic analysis of
tryptic peptides. Considerable charge heterogeneity was observed,
particularly for the -COP and
-COP subunits. The multiple
species detected, however, did not appear to reflect the presence of
distinct translation products but rather a significant degree of
protein phosphorylation. The observed pI of
-COP was sensitive to
alkaline phosphatase digestion. Moreover, isolation of coatomer from
metabolically labeled tissue culture cells demonstrated directly that
both
-COP and
-COP, but no other coatomer subunits, were
serine-phosphorylated. COPI phosphorylation may regulate coatomer
assembly, membrane recruitment, or the specificity of
coatomer-organelle interaction.
Transport of proteins between organelles is dependent on the
formation of vesicular carriers. Often, these transport vesicles are
formed in conjunction with the assembly of specific membrane-associated
coat proteins derived from cytosolic precursors. At least three types
of coat proteins are known, each associated with one or more transport
steps. Clathrin coats act to selectively accumulate plasma membrane
receptors at clathrin-coated pits, resulting in receptor endocytosis (1) and the transport of proteins from the trans-Golgi network
(TGN) ()to endosomes and perhaps other
destinations(2) . In addition, two distinct coat complexes,
designated COPI and COPII, play important roles early in the secretory
pathway (3, 4, 5, 6) . Both COP
complexes are well known to mediate transport among the ER,
intermediate compartment, and/or the Golgi complex by facilitating the
formation of COP-coated vesicles at one or more of these three
sites(6, 7, 8, 9) . Unlike
clathrin-coated vesicles, it is less clear whether COPI and/or COPII
vesicles selectively accumulate their intended cargo at the time of
vesicle budding. However, like clathrin-coated vesicles, COPI vesicles
do mediate multiple transport steps, being active not only in transport
among ER, intermediate compartment, and the Golgi complex, but also
between Golgi cisternae (2, 3, 4) . The role
of COPI in transport between the ER and the Golgi complex, as well as
in endosome function, has been established by inhibition of transport
by microinjected anti-
-COP antibody (10) as well as by the
isolation of COPI mutants in yeast and Chinese hamster ovary
cells(8, 11) . There is also considerable evidence
from in vitro reconstitution experiments that at least COPI
coats are involved in transport through the Golgi stack(12) .
COPI is recruited to membranes as a complex derived from a cytosolic
precursor called coatomer(13) . The coatomer complex is a high
molecular weight heterooligomer composed of seven proteins, -COP
(160 kDa),
-COP (110 kDa),
`-COP (102 kDa),
-COP (98
kDa),
-COP (61 kDa),
-COP (35 kDa), and
-COP (20 kDa),
each present in equimolar amounts(13, 14) . Binding of
coatomer to membranes is dependent on adp-RIBOSYLATION FACTOR, a small
myristoylated GTPase that is thought to bind GTP upon interaction with
a membrane-associated guanine nucleotide exchange
factor(15, 16, 17) . Exchange of GDP for GTP
facilitates coatomer binding by an as yet unknown mechanism that is
sensitive to the drug brefeldin A(18) . Assembly of the COPI
complex is thought to drive vesicle budding(19) . COPI assembly
in the ER or Golgi may also provide for the selective inclusion in
Golgi-derived recycling vesicles of resident ER proteins bearing the
KKXX retrieval motif(20) .
Although COPI in
vivo is concentrated in the intermediate compartment and cis-Golgi(21) , immunolocalization experiments also
suggest that at least some COPI is also found elsewhere in the Golgi
stack including the TGN(22, 23) . Thus, COPI may play
a functional role at sites other than those between the ER and cis-Golgi. Evidence is accumulating that COPI components are
involved in the endocytic pathway, apparently at the level of
endosomes. A CHO cell line bearing a temperature-sensitive mutation in
-COP exhibits a defect in the recycling of low density lipoprotein
receptors internalized from the plasma membrane(11) . Recent
evidence also demonstrates that COPI components bind specifically to
endosomes in vitro and that antibodies directed against
-COP inhibit endosome function(24) .
How might a single
protein complex act at multiple intracellular sites? One possibility is
that the COPI proteins are structurally heterogeneous, with multiple
isoforms of coatomer proteins combining to produce functionally
distinct COPI complexes. Such heterogeneity might also be achieved by
differential post-translational modification of one or more COPI
subunits, with isoforms of COPI possibly associating with different
organelles. Since little was known about the structure and composition
of coatomer complex(es), we decided to determine whether individual
members of the coatomer complex exhibited any biochemical
heterogeneity. Indeed, a surprising degree of heterogeneity was
observed, particularly in the and
subunits. We also found
that both subunits are phosphorylated, suggesting that phosphorylation
may be responsible for at least some of these differences.
To prepare cell lysates, spinner culture cells were lysed
in 4 volumes of wash buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 0.5% Triton X-100) brought to 1% Triton X-100 with protease
inhibitors (2 µg/ml pepstatin A, antipain, aprotinin, and
leupeptin, 0.5 mM 1, 10 phenanthroline, 1 µM chymostatin, 1 mM phenylmethylsulfonyl fluoride) and
phosphatase inhibitors (100 µM NaVO, 20 mM sodium glycerophosphate, 10 mM NaF). The cell pellet was
agitated on ice for 15 min and then centrifuged at 10,000
g for 15 min. The supernatant was cleared by centrifugation at
100,000
g for 20 min. Supernatants were kept at
-70 °C for up to 1 week.
Cytosols were prepared from
labeled cell pellets without detergent by suspension of the pellet in 2
volumes of TEAS (10 mM trienthanolamine/acetic
acid, pH 7.3, 1 mM EDTA, 250 mM sucrose) followed by
disruption using a ball bearing cell homogenizer (0.2496-inch ball)
until >80% cell lysis was achieved (assayed by trypan blue
exclusion).
Figure 1:
Purification of coatomer from rat
liver. Purification of COP I was as described under ``Materials
and Methods'' with Mono-Q column as the final step. An SDS-PAGE
separation of the complex was performed and visualized with Coomassie
Blue. The large gel is 6.5% polyacrylamide. Inset is
12% polyacrylamide. Positions of individual COPs are indicated at the right based upon Western blot identification with COP-specific
antibodies. Hash marks indicate positions of molecular mass
standards (205, 116.5, 80, and 49.5 kDa from top to bottom). ` can be seen as a distinct band below the
/
band. Doublet of delta COP is a degradation artifact in
this particular sample, while the doublet of epsilon COP was seen in
all samples.
We next analyzed the purified coatomer by
high resolution two-dimensional IEF/SDS-PAGE. The first dimension was a
denaturing pH 5-9 IEF; the second dimension was an 8% SDS gel.
Proteins were visualized by silver stain (Fig. 2, right
panel). To identify which spots corresponded to which coatomer
subunit, a series of two-dimensional gels were run in pairs. One of
each pair was transferred onto an Immobilon-P membrane. These membranes
were stained with Coomassie Blue for accurate alignment and probed with
subunit-specific antibodies. A composite of Western blots is shown in
the left panel of Fig. 2. The gels used in this
composite were selected for clarity and thus differ slightly in the
amount of protein loaded from the silver-stained gel. There is also
some intergel variability. To definitively identify a silver-stained
spot, multiple pairs of silver-stained and Western blotted membranes
were compared. It was thus possible to identify spots corresponding to
almost all coatomer subunits with the exception of -COP, which
apparently entered the IEF gel poorly, if at all.
-COP was not
retained by the 6.5% gel used for SDS-PAGE. For some COPI species (e.g.
-COP), the Western blotted pattern was often
broader than that observed by silver stain; in this case
-COP was
identified by the M
and pI of the visualized spot
and the absence of any neighboring spots even when the silver stain was
overdeveloped. Several silver-stained spots were not reactive with any
COPI antibody and presumably represented contaminants in the
preparation.
Figure 2: Two dimensional IEF/SDS-PAGE reveals heterogeneity of individual coatomer subunits. Rat liver COP I purified as under ``Materials and Methods'' with Superose-6 column as a final step separated in a two-dimensional (IEF/SDS-PAGE) system, pH 5-7 in the first dimension and 8% SDS-PAGE in the second dimension. The left panel shows Western blots with COP-specific antibodies from a series of two-dimensional gels. Each was compared with a parallel silver-stained gel to identify the corresponding protein spot. A single silver-stained two-dimensional gel is shown in the right panel for comparison.
The two-dimensional gel analysis revealed considerable
heterogeneity of the - and
-COP spots.
-COP existed as a
series of spots at 110 kDa. Four to five separate species (pI
5.5-6.0) were consistently observed in multiple preparations of
COPI analyzed by multiple two-dimensional gels, suggesting they
reflected multiple
-COP isoforms rather than deamidation or
carbamylation in the denaturing IEF first dimension gel. Because COPI
was purified to near homogeneity before two-dimensional gel analysis,
it is unlikely that this is the result of a fortuitous cross-reactivity
with a similarly sized group of unrelated proteins. This result is
further confirmed by the finding of 3-4
-COP immunoreactive
species in two-dimensional gels of total human keratinocyte
extracts(29) .
-COP was consistently detected as three
distinct 61-kDa species (pI 6.5-7.0) by both Western blot and
silver stain (Fig. 2). As for
-COP, multiple
-COP
species were observed upon two-dimensional gel electrophoresis. They
were also widely spaced, suggesting the spots represented isoforms
rather than IEF artifacts.
-COP was poorly soluble in the first
dimension, resulting in a diminished spot in the silver-stained gel
(and on Coomassie-stained filters); material precipitated at the basic
end of the IEF gel was detected in the Western blot (Fig. 2, left edge of left panel). Nevertheless, sufficient
-COP did enter the gel to be identified as pI
5.9 slightly
smaller than
-COP by both Western blot and silver stain.
Occasionally, immunoreactive
-COP appeared as a streak or two
partially resolved spots (Fig. 2), but this result was
inconsistent relative to the behavior of
- and
-COP.
-COP corresponded to a 36-kDa doublet or triplet (pI
4.6)
by silver stain. This was consistent with a recent report of partial
truncation of the
-COP amino terminus(30) . Identification
of
-COP was made primarily on the basis of M
and calculated pI. Western blots for
-COP typically revealed
a much broader streak. The reason for this disparity is unclear.
An
unknown 90-kDa protein (pI 5.2) was consistently found associated
with even the most homogenous preparations of coatomer (Fig. 2, right panel, arrow). It was associated with a smaller
spot of the same M
but a pI of
5.1. The
identity of these spots may be of interest due to their clear
propensity to be enriched through all stages of the coatomer
purification. Other contaminants observed between 40 and 55 kDa varied
between coatomer preparations.
Although the charge heterogeneity of
immunoreactive - and
-COP in highly purified coatomer
preparations might have reflected multiple
- and
-COP gene
products, further analysis strongly suggested this was not the case.
Purified coatomer was separated on preparative two-dimensional gels,
transferred to Immobilon-P membranes, and visualized with Coomassie
Blue. Each of the multiple
and
spots were separately
excised and subjected to tryptic digestion. Digested peptides were
analyzed by mass spectroscopy to generate a tryptic peptide map. All
large peptides obtained from each of the middle three (of five)
spots had identical sizes, strongly suggesting that they did not arise
from different gene products (not shown). The two most acidic
-COP
spots were also subjected to mass spectroscopy peptide analysis. As
with
-COP, a comparison of the 20 largest tryptic fragments from
each spot was identical, suggesting that each set of peptides arose
from the same protein and that modifications of the protein could not
be detected among the larger peptide fragments.
We also attempted to
identify closely related -COP cDNAs in a rat liver cDNA library by
probing with authentic
-COP cDNA probes at low stringency.
Polymerase chain reaction was also performed using a variety of
oligonucleotide primers corresponding to regions of high conservation
between rat and yeast
-COP sequences(23, 31) .
Both approaches yielded only multiple cDNA clones identical to
-COP. (
)Taken together, these negative results
suggested that the observed heterogeneity of
-COP, and possibly of
-COP, represented post-translational modification of both
proteins.
Phosphorylation of -COP-We first determined
whether
-COP in purified coatomer was phosphorylated. COPI
partially purified from rat liver was treated with alkaline phosphatase
at various concentrations and times and analyzed by two-dimensional
IEF/SDS-PAGE. Because the alkaline phosphatase preparations contained
contaminants of similar molecular weight,
-COP was identified by
Western blot. As shown in Fig. 3, alkaline phosphatase treatment
shifted the
-COP spots from their characteristic positions
(between pH 5.5 and 6.0) to the basic end of the gel (pH 9), suggesting
that dephosphorylation had occurred. A similar shift was also observed
for the corresponding silver-stained spots, although not for spots
corresponding to
-COP or
-COP; the
-COP spot was
obscured by the alkaline phosphatase itself (not shown). The shift of
immunoreactive
-COP was also dependent on Mg
and
was inhibited by EDTA, indicating that the shift was the result of
alkaline phosphatase activity and not proteolysis.
Figure 3:
-COP pI is shifted by alkaline
phosphatase treatment. Rat liver COPI (100 µg) was treated with
various concentrations of alkaline phosphatase for 1 h and separated on
two-dimensional gel as in Fig. 2, except that the first
dimension was pH 5-9. The second dimension was blotted onto
nitrocellulose and probed with EAGE for
-COP. Dephosphorylated
-COP appears at the base end of the gel. Lane 1, no added
alkaline phosphatase; lane 2, 3.36 µg/ml alkaline
phosphatase added; lane 3, 33.6 µg/ml alkaline phosphatase
added.
The magnitude of
the pI shift was greater than that expected for removal of a single
phosphate group. In addition, the total amount of immunoreactive (and
silver-stained) -COP also appeared to decrease, suggesting that
the dephosphorylation reduced the ability of the protein to enter the
IEF gel, perhaps by altering its solubility characteristics. For these
reasons, we sought to demonstrate directly that
-COP was
phosphorylated.
CHO cells were labeled for 12 h with
[P]orthophosphate and lysed in 1% Triton X-100
or homogenized in TEAS
. Labeled lysates were
immunoprecipitated with the anti-
-COP peptide antibody
E-1(23) , separated by 6.5 and 10% SDS-PAGE, and blotted onto
polyvinylidene difluoride filters. The filters were first
autoradiographed (Fig. 4, left two lanes of each panel). A single labeled band of
110 kDa was observed in
the immunoprecipitate but not in a control precipitation using only
protein A-Sepharose beads. To confirm that the labeled protein was
indeed
-COP, the filter was probed with a second mouse monoclonal
antibody against
-COP (M3A5)(32) . The same filter was
stripped and reprobed with a rabbit antibody directed against
`-COP. The
P-labeled band comigrated with
-COP
but not with
`-COP.
-COP was also detected in the
immunoprecipitate (Fig. 4, right panel, right
lane), but no corresponding
P-labeled band was
detected in this position. Thus, neither the co-precipitated
` nor
-COP were phosphorylated in CHO cells. Western blots using
antibodies directed against
-,
-,
-, and
-COPs were
negative (not shown), suggesting that the conditions used for
precipitation led to the partial dissociation of the coatomer into
smaller complexes, one of which contained
-,
`-, and
-COP. Elimination of detergent from the immunoprecipitation did
not enhance recovery of
-COP, suggesting that dissociation of the
COP complex may have been due to salt or freeze/thawing of COP samples
prior to immunoprecipitation. Interestingly, partial coatomer complexes
are also recovered under high salt conditions, although in this case
-COP associated with
and
` but not
-COP(25) .
Figure 4:
Immunoprecipitation of phosphorylated
-COP from CHO cell lysates. Lysates from CHO cells, metabolically
labeled with
P, were immunoprecipitated with the
anti-
-COP antibody E1 (+) or with protein A-Sepharose
alone(-). The precipitate was run on a 6.5% (left panel)
or 10% (right panel) SDS-PAGE and analyzed by autoradiography (32P) and then by sequential Western blots (
,
`, and
lanes). The Western blots were first visualized with the
monoclonal antibody M3A5 directed against
-COP. The same blots
were stripped and reprobed with rabbit antibodies directed against
` or
-COPs as indicated in each lane. Positions of
detected COPs are indicated. Positions of molecular mass standards are
indicated.
Figure 5:
Isolated coatomer contains phosphorylated
-COP and
-COP. A, COPI from CHO cells metabolically
labeled with
P was copurified with unlabeled rat liver
COPI. The resulting purified COPI was separated by SDS-PAGE and
autoradiographed (32P). A parallel lane containing whole CHO
cytosol was analyzed by Western blot to locate
-COP (upper
left panel) and
-COP (lower left panel). Positions
of
- and
-COPs are indicated, respectively, by upper and lower arrows at right. Bands appearing in
the lower left panel below 50 kDa were degradation products
developed in whole cytosol only. Hash marks indicate positions
of molecular mass standards (from top to bottom: 205,
116.5, 80, and 49.5 kDa). B, CHO cells labeled with
[
S]Met/Cys treated as above. Purified coatomer
was separated on 8% (upper panels) and 12% (lower
panels) SDS-PAGE, transferred to Immobilon-P, stained, and
autoradiographed. All COPI subunits (labeled at right) were
visible by both Coomassie Blue stain (left) and
autoradiography (right).
The fact that coatomer complexes
at least partly dissociated upon immunoprecipitation made it necessary
to provide additional evidence that none of the subunits had been
partially lost during purification: conditions similar to those used
for immunoprecipitation were used to purify coatomer from P-labeled CHO cells. To confirm that each of the COPI
subunits were recovered from CHO lysates, coatomer was purified from
[
S]methionine/cysteine-labeled cells in the
presence of excess unlabeled purified rat liver coatomer. As shown in Fig. 5B, all of the subunits were detected as
S-labeled bands comigrating with each of the seven
unlabeled rat liver COP components. The presence of each COP component
was also confirmed by Western blot analysis using subunit-specific
antibodies (not shown). The copurified material thus contained all COP
components. As phosphorylation was detected only on the
- and
-COPs, it is likely that only these components were
phosphorylated, although
-COP appeared to be phosphorylated more
heavily.
Finally, to determine whether -COP phosphorylation
occurred on serine, threonine, or tyrosine residues,
-COP was
immunoprecipitated from lysates of
P-labeled HeLa cells
and subjected to phosphoamino acid analysis. As shown in Fig. 6,
only phosphoserine was detected, indicating that at least
-COP was
phosphorylated only on serines.
Figure 6:
Phosphorylation of -COP is on serine.
Partially purified COP I from HeLa cells metabolically labeled with
P was separated by SDS-PAGE. The labeled band with an M
of 110,000 was eluted and subjected to mild acid
hydrolysis. The products were separated by thin layer electrophoresis
first at pH 1.9 and then in a second dimension at pH 3.5. Left
panel, authentic unlabeled phosphoserine (S),
phosphothreonine (T), and phosphotyrosine (Y)
standards were visualized with ninhydrin. Right panel,
autoradiograph of labeled hydrolyzed
sample.
We have found that several of the COPI subunits of the
cytosolic coatomer complex exhibit significant structural
heterogeneity. This heterogeneity is most obvious for -COP and
-COP;
-COP exists as three to five distinct species, while
-COP exists as up to three species with differing pI but identical
molecular weights. Because both
and
-COP are phosphorylated,
the simplest conclusion would be that differential phosphorylation of
- and
-COP is responsible for at least part of the observed
heterogeneity. Despite much effort, we have thus far been unable to
detect novel cDNAs or transcripts encoding distinct proteins closely
related to
-COP. Using antibody screening of expression libraries,
low stringency hybridization, and polymerase chain reaction, only
``authentic''
-COP cDNAs have been recovered. Similarly,
closely related proteins have yet to emerge in the sequence data base.
Thus it seems less likely that the existence of multiple
-COP
species reflects the existence of different gene products each
sufficiently related so as to cross-react with a common set of
anti-peptide antibodies.
Given the magnitude of the shift in pI upon
dephosphorylation of -COP with alkaline phosphatase, it was
impossible to determine the actual number of phosphates that exist per
-COP or to establish that the phosphorylation is completely
responsible for the heterogeneity observed after IEF/SDS-PAGE. Similar
considerations apply to
-COP. However, taking into account the
presence of five
-COP and three
-COP spots and noting that
- and
-COP all exist in the coatomer in roughly equimolar
amounts (by Coomassie Blue staining), the 3-4:1 ratio of
-COP:
-COP
P-labeling suggests that there may be
1-5 phosphorylations/
-COP molecule and 0-2
phosphorylations/
-COP molecule. The different levels of
phosphorylation are unlikely to be the result of differential turnover
rates, as the ratio was preserved whether labeling was for 4 or 12 h.
What role then may phosphorylation of COPI components play?
Phosphorylation could regulate the partitioning of COPI between
cytosolic and membrane-bound states. This possibility has at least one
precedent in the binding of the small GTPase Rab4 to endosomes. In this
case, phosphorylation of rab4 by p34 kinase leads to its
cytosolic accumulation in mitotic cells(33) .
Alternatively,
COPI phosphorylation may control the specificity of organelle binding.
It is becoming increasingly apparent that one or more COPI components
bind to and/or function at multiple intracellular sites in both the
secretory and endocytic
pathways(4, 11, 12, 20) .
Recruitment of COPI subunits to these various sites of action may, at
least in part, be controlled by the phosphorylation state of -COP
or
-COP. Consistent with this view,
-COP along with several
other COPI subunits is recruited to endosomes but not
- or
-COP(24) .
- and
-COP may either be dissociated
from a putative
-,
`-,
-COP complex before or after
binding; or they may be replaced in the holocomplex by novel
non-cross-reactive isoforms. Conceivably, phosphorylation may, under
certain conditions, help generate subcomplexes of coatomer. This
possibility is consistent with our finding that antibodies to
-COP
co-precipitated with
`- and
-COP but not with
-,
-,
-, or
-COP. In this case, antibody binding itself may cause a
partial dissociation of the complex. Binding to the cognate COPI
receptor on endosomes may have a similar effect.
Another intriguing
possibility is suggested by a variety of observations indicating that
the binding of coatomer, as well as membrane protein sorting and
vesicle budding, are events that are regulated by molecules associated
with signal transduction. Evidence is accumulating that G protein
activators can enhance and/or alter specific steps in intracellular
transport. Among these examples is the increase in ER to Golgi
transport (34) and transport from the TGN or endosomes to the
apical surface in polarized Madin-Darby canine kidney cells (35, 36) ()due to treatment with
AlF
, and cholera toxin. It is therefore interesting to note
that AlF4 and mastoparan enhance COPI binding at least to ER/Golgi
membranes, while conversely, the addition of G
subunits inhibits binding(7) . G protein activators do
not, however, enhance COPI binding to endosomes(24) .
Nevertheless, COPI does interact with the inositol phosphates inositol
1,3,4,5-tetrakisphosphate and inositol hexakisphosphate(37) .
Finally, binding of ADP-ribosylation factor to membranes activates
phospholipase D activity, an event that is likely to directly or
indirectly affect COPI recruitment(16) .
Phosphorylation of
COPI subunits is a link between G protein enhancement of coatomer
binding and transport. Conceivably, this link would be provided if COPI
subunits were substrates for protein kinase C. Phorbol esters (direct
activators of protein kinase C) activate transport between the ER and
the Golgi (a COPI-mediated process)(34) , and binding of both
ADP-ribosylation factor and -COP is regulated by protein kinase C
activity in rat basophilic leukemia cells(38) . Although we
have not identified the specific residue(s) phosphorylated in
-COP, there are three consensus protein kinase C serine
phosphorylation sites in the rat
-COP sequence
(K
SVK, K
KES and K
KTS). Other
sites for protein kinase C or other kinases whose activities are
regulated by signal transduction events may also exist. Differential
-COP phosphorylation in response to different signals may regulate
the recruitment of COPI to specific intracellular sites in response to
shifts in demand to vesicular traffic between compartments. This idea
is also appealing in light of the behavior of protein kinase C itself
in response to activation. Inactive protein kinase C is cytosolic,
whereas activated protein kinase C becomes
membrane-bound(39, 40) . Indeed, some activated
isoforms of protein kinase C have been found to bind directly to Golgi
membranes(41) . Conceivably, localization of activated protein
kinase C may help define sites of vesicle budding by phosphorylating
and locally recruiting coatomer complexes. These considerations each
create a number of highly testable possibilities.