(Received for publication, January 18, 1996)
From the
At the nonpermissive temperature of 39.5 °C, the Chinese
hamster ovary cell conditionally lethal, temperature-sensitive (ts)
mutant ldlF exhibits the following defects: rapid degradation of low
density lipoprotein receptors, disruption of ER-through-Golgi
transport, and disintegration of the Golgi apparatus. All of these are
corrected by transfection with an expression vector for wild-type
-COP, a subunit of coatomers (Guo, Q., Vasile, E., and Krieger,
M.(1994) J. Cell Biol. 125, 1213-1224). We now report
the identification in ldlF cells of a point mutation in the
-COP
gene, Glu
to Lys
, which prevents the
corresponding cDNA from correcting the defects in transfected ldlF
cells and the immunochemical analysis of the synthesis, structure, and
stability of
-COP. At the permissive temperature (34 °C), the
steady state level of ts-
-COP in ldlF cells was about half that of
-COP in wild-type Chinese hamster ovary cells and the isoelectric
point of ts-
-COP was 0.14 pH units higher than that of the
wild-type protein. The stability but not the biosynthesis of
ts-
-COP was temperature-sensitive (t
> 6
h at 34 °C and
1-2 h at 39.5 °C), and this accounts
for the virtual absence of detectable ts-
-COP protein in ldlF
cells after incubation at 39.5 °C for >6 h. The steady state
levels in ldlF cells of another coatomer subunit,
-COP, and the
peripheral Golgi protein ldlCp were not temperature-sensitive. Thus, a
mutation in
-COP that causes instability at 39.5 °C is
responsible for all of the temperature-sensitive defects in ldlF cells,
and the stability of
-COP is not linked directly to that of
-COP. ldlF cells should be useful for the future analysis of the
structure and function of
-COP, the assembly of COPs into
coatomers, and the participation of coatomers in intracellular membrane
transport.
The endocytic and secretory pathways of intracellular membrane traffic appear to depend on multisubunit protein complexes (e.g. coatomers, NSF/SNAPs/SNAREs) to catalyze and regulate the membrane fusions and fissions required for transport (Bennett and Scheller, 1993; Rothman, 1994; Pryer et al., 1992; Warren, 1993). At least some of the components of these complexes participate in reactions used throughout the secretory and endocytic pathways.
In
the course of mammalian somatic cell genetic analysis of membrane
traffic, we isolated and characterized a conditionally lethal, ts ()Chinese hamster ovary (CHO) cell mutant, called ldlF,
which behaves normally at the permissive temperature (34 °C) but
exhibits pleiotropic defects at the nonpermissive temperature (39.5
°C) (Hobbie et al., 1994; Guo et al., 1994).
These include rapid degradation of LDL receptors, presumably due to
missorting, disruption of ER-through-Golgi transport of integral
membrane and secreted proteins, and disintegration of the Golgi
apparatus in a manner that resembles the effects of the drug brefeldin
A on wild-type CHO cells (Takatsuki and Tamura, 1985; Fujiwara et
al., 1988; Lippincott-Schwartz et al., 1989, 1990; Shite et al., 1990; Orci et al., 1991). We cloned from a
CHO cell cDNA expression library a cDNA that, when transfected into
ldlF cells, corrected all of their ts pleiotropic defects in membrane
transport (Guo et al., 1994). The predicted sequence of the
protein encoded by this cDNA is virtually identical to that of bovine
-COP, which was cloned contemporaneously (Hara-Kuge et
al., 1994).
-COP is one of seven coat proteins (
,
,
`,
,
,
, and
) that form a stable
complex called the coatomer COPI. COPI is distinct from a second coat
complex, called COPII, which has also been implicated in membrane
traffic (Barlowe et al., 1994; Bednarek et al.,
1995). COPI coatomers can be found in the cytoplasm and associated with
the membranes of the Golgi apparatus, the ER or nonclathrin-coated
(COP-coated) vesicles (Duden et al., 1991a; Waters et
al., 1991; Serafini et al., 1991; Stenbeck et
al., 1992, 1993; Pepperkok et al., 1993; Ostermann et
al., 1993; Orci et al., 1994). They have been shown to be
required for the formation of functional Golgi transport vesicles in vitro (Ostermann et al., 1993), and they may be
involved in anterograde (ER to trans Golgi) (Bednarek et al.,
1995) and retrograde (Golgi to ER) vesicular transport (Letourneur et al., 1994) and possibly in endosome function (Whitney et al., 1995).
The correction of the ts defects of ldlF
cells by transfection with an -COP cDNA suggested that these cells
might carry a mutation in the
-COP gene itself. However,
the data did not rule out the possibility that
-COP was
functioning as an extragenic suppressor and that a mutation in some
other gene in ldlF cells was responsible for their ts phenotypes. Here
we show that there is a point mutation in the
-COP gene in ldlF
cells (ts
-COP). The mutation converts Glu
to
Lys
, prevents the corresponding cDNA from correcting the
defects in transfected ldlF cells, and results in the destabilization
of the ts-
-COP protein at the nonpermissive temperature. Thus,
ldlF cells provide direct genetic evidence that in animal cells
-COP, and thus the COPI coatomer complex (Kuge et al.,
1993), is essential for establishing or maintaining Golgi structure and
is required either directly or indirectly for both ER-through-Golgi
transport and normal endocytic recycling of LDL receptors.
Bacterial colonies from a cDNA library made
from ldlF were screened by hybridization according to standard
techniques (Sambrook et al., 1989). A 0.95-kb fragment of
-COP cDNA was used as a probe. The probe DNA was labeled with
[
-
P]dATP using the megaprime DNA labeling
system (Amersham Corp.). Hybridization was carried out at high
stringency. In brief, after hybridization overnight at 61 °C with
hybridization buffer (500 mM sodium phosphate buffer, pH 7.0,
containing 1 mM EDTA, 7% SDS, 1% bovine serum albumin, and 100
µg/ml salmon sperm DNA), the filters were washed three times at
room temperature with 300 mM phosphate buffer, two times at 60
°C with wash solution A (150 mM phosphate buffer, 5% SDS,
0.5% bovine serum albumin, 1 mM EDTA), and two times at 60
°C with wash solution B (150 mM phosphate buffer, 1% SDS,
1 mM EDTA). One positive clone was isolated after screening
53,640 colonies, and two colonies were isolated after a second
screening. The plasmids (designated pts-
-COPx) containing
cDNAs from the ldlF cells along with the plasmid (p
-COP,
formerly called pLDLF-1 (Guo et al., 1994))
containing the previously isolated cDNA for wild-type CHO
-COP
were subjected to side-by-side DNA sequence analysis. Sequences were
determined at least once on both strands using the dideoxy chain
termination method with Sequenase (U. S. Biochemical Corp.). Two
differences between p
-COP and pts-
-COPx were noted, a point mutation at base 751 within a 0.3-kb PstI-BstEII fragment in the coding sequence and
pts-
-COPx contained an extra 11 base pairs before the
poly(A) tail. The predicted isoelectric points of the
-COP and
ts-
-COP proteins were calculated using the program ISOELECTRIC
(Genetics Computer Group Sequence Analysis Software Package, version
7.3, Madison, WI; Devereux et al. (1984)).
The 0.3-kb PstI-BstEII fragment of the p-COP was
replaced with the corresponding altered fragment from
pts
-COPx. The resulting plasmid pts-
-COP differs from the wild-type p
-COP by only a single
point mutation at base 751. In transfection assays in which the
activities of pts-
-COP and pts-
-COPx were
compared, there were no differences observed between these plasmids
(see ``Results'' and data not shown). Thus, the extra 11 base
pairs in pts-
-COPx are not functionally relevant.
Cytosols were prepared essentially as described by Spiro et al.(1995). Cells grown as described above were released
from the culture dishes by mild trypsinization, collected by
centrifugation (5.0 min at 250 g at 4 °C), and
washed three times with ice-cold PBS. Pellets were suspended in
ice-cold breaking buffer (20 mM Hepes, pH 7.4, containing 0.10 M KCl, 85 mM sucrose, 20 mM EGTA) and were
homogenized in a stainless steel ball homogenizer until 80-90% of
the cells were disrupted. Post-nuclear supernatants were prepared by
centrifugation at 800
g for 5 min at 4 °C. They
were subjected to centrifugation at 380,000
g for 15
min at 4 °C to yield cytosols. The protein concentrations of the
whole cell extracts and cytosols were determined using the DC Protein
Assay kit from Bio-Rad Laboratories. The whole cell extracts and
cytosols were stored at -150 °C prior to use.
For detection of radioactively labeled proteins, gels
were treated with Amplify fluorography reagent (Amersham Corp.), dried,
and exposed to Kodak XAR-5 x-ray film at -80 °C. For
immunoblot analysis, proteins in the gels were electrophoretically
transferred to nitrocellulose membranes (Schleicher & Schuell; pore
size, 0.22 µm) as described by Towbin et al.(1979). The
membranes were blocked overnight at 4 °C with 5% blocking reagent
from the ECL detection system (Amersham Corp.) in PBS containing 0.1%
Tween 20 (PBS-T). Blocked membranes were probed with primary antibody
(-COP: 1 µg/ml of anti-
-COP; ldlCp: 3 µg/ml of
anti-Cpep (Podos et al., 1994);
-COP: 1:100 dilution of
T. Kreis's monoclonal antibody M3A5, a gift from R. Klausner and
J. Donaldson) in PBS-T for 1 h at room temperature, then incubated with
horseradish peroxidase-conjugated anti-rabbit (for anti-
-COP and
anti-Cpep) or anti-mouse (for M3A5) antibody in PBS-T for 1 h at room
temperature, and developed using the ECL detection system following the
manufacturer's instructions.
We have previously shown that transfection with -COP cDNA corrects all of the temperature-sensitive defects in membrane
trafficking in the CHO cell conditional lethal mutant ldlF (Guo et
al., 1994). This result suggested that the primary defect in ldlF
cells was a mutation in the
-COP gene. However, the data
did not rule out the possibility that
-COP was functioning as an
extragenic suppressor and that some other defective gene in ldlF cells
was responsible for their ts phenotypes. We directly addressed this
question by cloning the
-COP gene from ldlF cells (see
``Experimental Procedures''). From a single colony giving a
strong hybridization signal, we isolated two clones. Both strands of
the entire cDNA inserts of these two clones were sequenced and compared
with the sequence of the wild-type
-COP. Fig. 1A shows the only portion of the coding region in
which the sequences of the wild-type (wt) and mutant (ts) cDNAs differed. A single point mutation in the mutant
sequence (C-to-T in the antisense strand shown in Fig. 1A) was observed. This substitutes Lys
in the mutant for Glu
in the wild-type protein (see Fig. 1A) and changes its net charge by two at
physiologic pH. This mutation in ldlF cells was further confirmed by
direct sequencing of polymerase chain reaction products amplified from
genomic DNAs and from two independent cDNA libraries (data not shown).
Unexpectedly, this mutant sequence was the only sequence of
-COP
present in either the genomic DNA or cDNAs, suggesting that ldlF cells
contain only one mutant allele at the
-COP locus.
Figure 1:
Comparisons of the sequences
of -COP cDNAs (antisense strands) from wild-type CHO (wt)
and ldlF (ts) cells and the isoelectric points of the
corresponding proteins. A, sequence analysis. The sequences of
the antisense strands of
-COP cDNAs from wild-type CHO cells (wt, single specimen, left lanes) and ldlF mutant
cells (ts, two specimens, right lanes) were
determined as described under ``Experimental Procedures.''
The base triplet for amino acid 251 is indicated. Base 751 (bold), a C in wild-type CHO, is mutated to T in ldlF. This
results in the substitution of Lys for Glu at residue 251. B,
isoelectric focusing. Cytosols (10 µg of protein/lane) prepared
from CHO (left lane) or ldlF (center lane) cells
grown at 34 °C and a 1:1 mixture of the two (right lane)
were subjected to isoelectric focusing followed by immunoblot analysis
using an anti-
-COP antibody and an ECL detection system as
described under ``Experimental Procedures.'' Focusing was
from the basic to the acidic end as indicated, and the pH gradient was
measured as described under ``Experimental Procedures.'' The
signals could be competed away by the presence of an excess of the
synthetic peptide against which the anti-
-COP antibody was raised
(not shown).
To confirm
the presence of this mutation in the protein product of the mutant
-COP gene in ldlF cells, we prepared a rabbit polyclonal
antipeptide antibody (designated anti-
-COP) directed against the
carboxyl terminus of hamster
-COP (see ``Experimental
Procedures'') and used immunoblotting of isoelectrically focused
samples to compare the
-COP proteins in CHO and ldlF cells grown
at the permissive temperature (Fig. 1B). The cells each
expressed only one form of the protein:
-COP in CHO cells
(apparent pI of 5.30 in this system); ts-
-COP in ldlF cells
(apparent pI of 5.44). The observed difference in the apparent
isoelectric points, 0.14 pH units, was virtually identical to that
predicted from the protein sequences (0.15 units). The absolute pI
values were somewhat higher than predicted (
0.45 units); this was
presumably due to the nature of the isoelectric focusing system used.
The presence of a single, abnormally basic, form of
-COP in ldlF
cells (ts-
-COP) is consistent with the observation of a single
mutant allele of the
-COP gene determined by cDNA and genomic
sequencing.
To determine if the substitution of Lys for Glu interfered with
-COP function, we constructed an expression
vector, pts-
-COP, which contains this single point
mutation, and examined its ability to correct the temperature-sensitive
lethality of ldlF cells. Wild-type (p
-COP) or mutant
(pts-
-COP) expression vectors were cotransfected with
pSV2neo into ldlF cells. The survival of G418-resistant colonies after
14 days of incubation at either the permissive temperature (34 °C,
a measure of transfection efficiency) or the nonpermissive temperature
(39.5 °C, a measure of
-COP function) was assessed by staining
fixed cells with crystal violet and visual inspection. Fig. 2shows that although the transfection frequencies were
similar for the two plasmids (34 °C, left panels), the
mutant was far less efficient in correcting the temperature-sensitive
lethality of ldlF cells than was the wild-type (39.5 °C, right
panels). Whereas many wild-type cDNA transfected colonies survived
at 39.5 °C (Fig. 2, upper right), only a few
mutant-transfected colonies survived the initial selection at 39.5
°C (lower right), and only three out of eight that were
tested could survive passage to mass culture (see below for further
discussion of these unusual colonies). No colonies survived at 39.5
°C when p
-COP was substituted by the corresponding
vector control containing no cDNA insert (data not shown). We conclude
that the Glu
to Lys
mutation disrupts
-COP function at the nonpermissive temperature and that this is
the mutation responsible for all of the temperature-sensitive defects
in membrane traffic in ldlF cells.
Figure 2:
Temperature-sensitive growth of ldlF cells
transfected with wild-type and mutant -COP cDNAs. On day 0, the
ldlF cells were plated at 34 °C. On day 2, the cells were
cotransfected with a mixture of either p
-COP or
pts-
-COP with pSV2neo (see ``Experimental
Procedures''). The cells were subsequently incubated in medium E
containing 175 µg/ml G418 either at 34 °C for 14 days to
monitor transfection efficiency (left panels) or at 39.5
°C for 14 days to test for reversion of their ts lethal phenotypes (right panels). Cells incubated at 39.5 °C were plated at
a density double that of those incubated at 34 °C (see
``Experimental Procedures''). Surviving colonies were
visualized by staining with crystal violet.
The biochemical consequences of
the Glu to Lys
mutation in
-COP were
characterized using the anti-
-COP antibody. In Fig. 3, CHO
and ldlF cells were preincubated at the indicated temperatures for 12
h, then pulse-labeled at the same temperatures for 30 min with
[
S]methionine, and lysed, and the lysates were
subjected to immunoprecipitation, SDS-polyacrylamide gel
electrophoresis, and autoradiography as described under
``Experimental Procedures.'' Although three major bands were
observed in the immunoprecipitate from the CHO cells at 34 °C (lane 1), the immunoprecipitation of only one (
36 kDa,
-COP) was blocked by a large excess of the synthetic
peptide against which anti-
-COP was raised (lane 2). In
immunoblot analysis of proteins, only a single band of
36 kDa was
recognized by anti-
-COP (data not shown). Because the mass of this
protein is similar to that predicted from the primary sequence of
-COP, 34.5 kDa (Guo et al., 1994), and because its
immunoprecipitation by anti-
-COP could be blocked by the synthetic
peptide, we conclude that this protein is hamster
-COP and that
the other two bands in the immunoprecipitates represent nonspecific
precipitation background. One of these, labeled x, provided a
useful control in experiments described below.
Figure 3:
Immunoprecipitation of wild-type and
ts-mutant -COPs from metabolically labeled wild-type CHO and ldlF
cells. On day 0, cells were plated in 6-well dishes at 150,000
cells/well in medium D at 34 °C. On day 1, cells were either
shifted to 39.5 °C or were maintained at 34 °C for an
additional 12 h as indicated. On day 2, the cells were pulse labeled
with [
S]methionine (400 µCi/ml) in
methionine-free medium F for 30 min, washed once with Ham's F-12
medium, and lysed, and the lysates were immunoprecipitated with
anti-
-COP in the absence (lanes 1 and 3-6)
or presence (lane 2) of 50 µg of the peptide to which
anti-
-COP was raised. The immunoprecipitates were reduced and
analyzed by 10% SDS-polyacrylamide gel electrophoresis and
autoradiography.
-COP from wild-type CHO cells (lanes
1-4) and ts-
-COP from mutant ldlF cells (lanes 5 and 6) exhibit identical electrophoretic mobilities in
this system (arrows). The band labeled x represents a
protein of unknown identity.
Fig. 3also
shows that the electrophoretic mobility in SDS-polyacrylamide gels of
the mutant form of -COP in ldlF cells, ts-
-COP (lanes 5 and 6), was indistinguishable from that of wild-type
-COP (lanes 1, 3, and 4). Strikingly,
the rate of synthesis of either
-COP in CHO cells or ts-
-COP
in ldlF cells was not temperature-sensitive (compare intensities of the
bands in lanes 3 and 4 with those in lanes 5 and 6). Thus, substantially reduced synthesis of
-COP at the nonpermissive temperature cannot account for the
defects in ldlF cells. Interestingly, at both 34 and 39.5 °C,
ts-
-COP in ldlF cells appeared to be synthesized at approximately
one-half the rate of
-COP in wild-type CHO cells. In addition,
quantitative immunoblot analysis (data not shown) was used to show that
for cells grown at 34 °C, the ratio of the steady state level of
-COP in CHO cells to that of ts-
-COP in ldlF cells was
2:1. These results are consistent with the possibility that
wild-type CHO cells express
-COP from two loci, whereas in ldlF
cells there is only a single, mutant locus for ts-
-COP.
Because
ts--COP was synthesized normally in ldlF cells, we examined the
possibility that the defects in ldlF cells were due to instability of
ts-
-COP at the nonpermissive temperature. We compared the rates of
degradation of newly synthesized
-COP in CHO cells at 34 and 39.5
°C with those of newly synthesized ts-
-COP in ldlF cells. Fig. 4A shows the results of an experiment in which
cells were pulse-labeled for 30 min with
[
S]methionine at 34 °C and then chased in
medium containing unlabeled methionine for the indicated times at
either 34 or 39.5 °C, prior to immunoprecipitation with
anti-
-COP, electrophoresis, and autoradiography. In wild-type CHO
cells at 34 °C, (Fig. 4A, top panel, left),
-COP was relatively stable and readily detected
throughout the 6-h chase at comparable levels at both 34 and 39.5
°C. Similar results were observed for
-COP at 39.5 °C (top panel, right) and for the background band x at both 34 and 39.5 °C. In ldlF cells at 34 °C (Fig. 4A, bottom panel, left),
ts-
-COP was almost as stable as
-COP in CHO cells. However,
in ldlF cells at 39.5 °C (bottom panel, right),
ts-
-COP was much less stable than
-COP in CHO cells. There
was a significant decrease in the intensity of the band after 1 h of
chase at 39.5 °C and very little signal after 3 h of chase. In
contrast, there was no significant temperature dependence of the
stability of background band x.
Figure 4:
Temperature dependence of the stabilities
of -COP, ts-
-COP,
-COP, and ldlCp in CHO and ldlF cells. A, immunoprecipitation analysis. On day 0, CHO and ldlF cells
were plated in 3 ml of medium D in 6-well dishes (150,000 cells/well)
at 34 °C. On day 2, the cells were pulse labeled with
[
S]methionine (350 µCi/ml) for 30 min at 34
°C and washed once with Ham's F-12 medium. The cells were
then lysed (time 0) or refed with medium D containing 1 mM unlabeled methionine prewarmed to 39.5 or 34 °C and chased at
39.5 or 34 °C for the indicated times. The cells were then lysed,
the lysates were subjected to immunoprecipitation with anti-
-COP,
and the precipitates were reduced and analyzed by 10%
SDS-polyacrylamide gel electrophoresis and autoradiography. The band
labeled x represents a protein of unknown identity. B, immunoblot analysis. On day 0, ldlF cells were plated in 5
ml of medium B in 60-mm dishes (570,000 cells/dish) at 34 °C. On
day 2, the cells were shifted to 39.5 °C for the indicated times,
then harvested, and lysed. The lysates were reduced and subjected to 8%
SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of a single
filter divided to permit separate analysis of high (>43 kDa) and low
(<43 kDa) molecular weight proteins was performed using either a
mixture of antibodies to
-COP and ldlCp (upper panel) or
-COP (lower panel) and the ECL detection system as
described under ``Experimental
Procedures.''
The results of the
immunoprecipitation experiments were confirmed by analysis of the
steady state levels of ts--COP using immunoblotting. Fig. 4B shows that after shifting ldlF cells to 39.5
°C, the cellular pool of ts-
-COP could be seen to drop after 1
h and was virtually completely depleted after 6 h (Fig. 4B, bottom panel). The rate of
degradation of ts-
-COP determined using immunoblotting (Fig. 4B, bottom panel) was somewhat lower
than that of newly synthesized ts-
-COP observed using
immunoprecipitation (Fig. 4A, bottom panel, right). To determine if the temperature-dependent instability
of ts-
-COP in ldlF cells was protein-specific, we examined the
steady state levels both of another coatomer subunit,
-COP, and of
an independent, brefeldin A-sensitive peripheral Golgi protein, ldlCp
(Podos et al., 1994). Neither
-COP nor ldlCp exhibited
significant temperature-dependent instability (Fig. 4B, top panel). Taken together, these results indicate that 1)
ts-
-COP is abnormally unstable at the nonpermissive temperature in
ldlF cells, 2) its temperature-dependent rapid degradation does not
represent a general destabilization of Golgi-associated proteins, and
3) the instability of ts-
-COP at the nonpermissive temperature is
apparently responsible for the pleiotropic ts defects in ldlF cells.
A single point mutation in the -COP gene in ldlF cells
results in the substitution of Lys
in the mutant
(ts-
-COP) for Glu
in the wild-type protein. This
mutation can account for the pleiotropic ts defects in ldlF cells,
including defects in membrane trafficking (secretion and LDL receptor
instability), disruption of the structure of the Golgi apparatus, and
instability of the ts-
-COP protein itself. The mutation in
ts-
-COP did not alter the stabilities of two other cytoplasmic,
peripheral Golgi proteins,
-COP (Duden et al., 1991a,
1991b) and ldlCp (Podos et al., 1994). Thus, the current work
supports the conclusion that ldlF cells provide the first genetic
evidence that
-COP and thus COPI coatomers in animal cells are
essential for establishing or maintaining Golgi structure and are
required either directly or indirectly for both ER-through-Golgi
transport and normal endocytic recycling of LDL receptors (Guo et
al. 1994). Furthermore, these findings suggest that ldlF cells
should be useful for studies of the relationship of
-COP's
structure to its function. Analysis of the effects of site-specific
mutagenesis on the ability of
-COP to correct the multifaceted
membrane transport defects in ldlF cells in vivo and in
vitro should help define the molecular mechanisms underlying
-COP's function.
In this regard, additional studies will
be required to define precisely how the Lys to
Glu
mutation affects the function and stability of
ts-
-COP. There was a very low but significant and reproducible
level of survival at 39.5 °C of ldlF cells transfected with
pts-
-COP. (Fig. 2, bottom right panel).
Using immunoblot analysis, we found that two out of three of the
surviving transfected colonies examined overexpressed ts-
-COP at
39.5 °C when compared with untransfected controls (data not shown).
The increased expression of ts-
-COP in these transfectants raises
the possibility that the mutant protein may be at least partially
functional and that overexpression provides a sufficient steady state
level of
-COP activity at 39.5 °C for the membrane traffic
required for survival and growth. We have previously observed in a
different mutant CHO cell line (ldlC) the suppression of a mutant
phenotype (LDL receptor deficiency) due to overexpression of an
unstable protein (abnormally glycosylated LDL receptor) (Reddy and
Krieger, 1989).
The molecular mechanism responsible for the thermal
instability of ts--COP is unknown. The mutation might directly
result in intrinsic temperature instability of the ts-
-COP
protein, regardless of its interactions with other cellular components.
Alternatively, the mutation might interfere with the incorporation of
ts-
-COP into coatomers (or some other complex) at the
nonpermissive temperature. The uncomplexed but perhaps otherwise normal
protein might then be subject to abnormally rapid degradation. To
address this issue, we attempted an immunochemical analysis of cells
that were expected to simultaneously express
-COP and ts-
-COP
at the permissive temperature by examining ldlF cells transfected with
the wild-type p
-COP cDNA (ldlF[LDLF]
cells; Guo et al., 1994). The normal phenotypes of
ldlF[LDLF] cells at the nonpermissive temperature
are due to the expression of wild-type
-COP (Guo et al. 1994). Using isoelectric focusing and either immunoprecipitation
or immunoblotting, we could readily detect
-COP but were unable to
detect significant levels of ts-
-COP in the cells grown at the
permissive temperature (data not shown). The mechanism
(transcriptional, translational, or post-translational) for the
suppression of ts-
-COP expression in the presence of
-COP
expression has not yet been determined. It is possible that the
incorporation of
-COP protein into coatomers was strongly favored
over that of ts-
-COP and that the unincorporated ts-
-COP was
unstable. Additional experiments will be required to resolve this
issue.
Because -COP is an integral component of COPI coatomers
(Hara-Kuge et al., 1994), ldlF cells may be helpful in
studying the assembly and functions of coatomers. Here we found that
the stability of at least one coatomer component,
-COP, was not
significantly altered in the absence of normal levels of
-COP.
This finding is similar to the observation by Duden et
al.(1994) that in Saccharomyces cerevisiae, depletion of
-COP (Sec26p) does ``not lead to the loss of other coatomer
subunits.'' Additional studies will be required to determine 1) if
the stabilities of other COPs are affected at the nonpermissive
temperature in ldlF cells, 2) if the intracellular distribution of the
other coatomer subunits is altered in the absence of
-COP, 3) if
stable coatomer-like complexes can assemble in the absence of
-COP, and 4) if such
-COP-deficient complexes can exhibit any
coatomer activity (e.g. ARF-dependent membrane attachment).