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(Received for publication, June 27, 1997, and in revised form, August 7, 1997)
From the Cellular Biochemistry and Biophysics Program, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021
ABSTRACT Members of the p24 family of putative cargo
receptors are proposed to contain retrograde and anterograde
trafficking signals in their cytoplasmic domain to facilitate coat
protein binding and cycling in the secretory pathway. We have analyzed
the role of the transmembrane domain (TMD) of a p24 protein isolated
from COPI-coated intra-Golgi transport vesicles. CD8-p24 chimeras were transiently expressed in COS7 cells and analyzed by immunofluorescence and pulse-chase experiments. The localization and transit of the wild-type chimera from the endoplasmic reticulum (ER) through the Golgi
complex involved a glutamic acid residue and a conserved glutamine in
the TMD. The TMD glutamic acid mediated the localization of the
chimeras to the ER in the absence of the conserved glutamine. Efficient
ER exit required the TMD glutamine and was further facilitated by a
pair of phenylalanine residues in the cytoplasmic tail. TMD residues of
p24 proteins may mediate the interaction with integral membrane
proteins of the vesicle budding machinery to ensure p24 packaging into
transport vesicles.
INTRODUCTION Transport in the secretory pathway is mediated by a vesicular
carrier mechanism that allows the cells to preserve organelle identity
by selectively packaging cargo molecules destined for secretion (1, 2).
Trafficking between the ER1
and the Golgi apparatus as well as intra-Golgi transport (1-4) employs
coatomer, a complex of seven subunit proteins (5). The COPI coat is
comprised of coatomer and the GTPase ADP-ribosylation factor (6, 7).
These two cytosolic proteins are sufficient to pinch off COPI-coated
vesicles from Golgi membranes in a cell-free reaction (8). Anterograde-
and retrograde-directed COPI-coated vesicles bud from every cisternae
of the Golgi complex in vivo (9).
The sorting of secreted proteins requires adaptor molecules to link
cargo to the cytoplasmic vesicle budding machinery. Cargo receptors,
possibly including p24 proteins (10-16) and VIP36-like lectins
(17-19), are proposed to move bidirectionally in the secretory pathway
to carry out their function (1, 2). The p24 proteins have previously
been identified as components of COPI- and COPII-coated vesicles in
mammals and yeast, respectively (12-14). They form a large family of
type I integral membrane proteins with a characteristically short
cytoplasmic tail, a predicted coiled-coil domain juxtaposed to the TMD
in the luminal/exoplasmic region, and a highly variable NH2-terminal domain (10-16, 20). Based on the analysis of
yeast strains lacking the p24 members Emp24p and/or Erv25p, it was
suggested that p24 proteins might be involved in the packaging of cargo molecules into vesicles as well as vesicle budding (13-15).
Furthermore, yeast strains lacking Emp24p were found to be defective in
the retention of ER-resident proteins (21) consistent with a role of
p24 proteins in controlling the fidelity of cargo recruitment into
budding vesicles.
We have previously shown that conserved phenylalanine residues in the
cytoplasmic domain of p24 proteins mediate the interaction with
coatomer and are likely to be involved in anterograde trafficking of
p24s from the ER through the Golgi complex (12, 20). As a first step
toward the identification of additional components of the vesicle
budding machinery we have further characterized the transport signals
of p24 proteins. A charged amino acid in the p24 TMD appears to serve
as a retention signal that is modulated by other TMD residues and
phenylalanines in the cytoplasmic domain.
EXPERIMENTAL PROCEDURES The OKT8 CD8 monoclonal antibody was obtained
from Ortho Diagnostic Systems (Raritan, NJ), the goat anti-mouse
fluorescein-conjugated secondary antibody from Molecular Probes
(Eugene, OR), and Lipofectin and LipofectAMINETM from Life
Technologies, Inc. COS7 (SV40-transformed African green monkey kidney)
cells were purchased from the American Type Culture Collection
(Rockville, MD), protein G-agarose from Boehringer Mannheim, and
[35S]methionine/cysteine from ICN.
The CD8 chimera were
constructed by the polymerase chain reaction such that the 165-amino
acids of the human CD8 extracellular domain were preserved (20). Codon
166 was changed to glycine to introduce a unique ApaI
restriction site, followed by a conserved proline, a stop codon, and a
EcoRI site (CD8-C1). Oligonucleotides (Gene Link, Thornwood,
NY) coding for the COOH-terminal 34 amino acids of chop24a
(RVVLWSFFEALVLVAMTLGQIYYLKRFFEVRRVV) and variants thereof
(see Fig. 1B), preceded by an ApaI site and
followed by a stop codon and EcoRI site, were annealed,
subcloned into the CD8 construct, and inserted into the pECE vector
(22). Sequences were verified by DNA sequencing using the Sequenase DNA
sequencing kit (U. S. Biochemical Corp.).
Transfection of COS7
cells was carried out with Lipofectin and LipofectAMINE for
immunofluorescence and pulse-chase analysis, respectively, according to
the manufacturer's instructions. The chimeras were analyzed 46 h
after transfection. For localization by immunofluorescence, the cells
were incubated in medium containing 10 µg/ml cycloheximide for 2 h prior to fixation. They were permeabilized with Triton X-100 before
labeling with the OKT8 CD8 monoclonal antibody (dilution of 1/50) and
goat anti-mouse fluorescein-conjugated secondary antibody (dilution of
1/200) as described (17) and viewed and photographed with an Axiophot
photomicroscope (Carl Zeiss, Oberkochen, Germany). For pulse-chase
analysis COS7 cells were labeled for 20 min with
[35S]methionine/cysteine and then incubated for 0, 15, 30, 45, 60, and 120 min in medium containing unlabeled
methionine/cysteine. The cells were lysed in Triton X-100, and the CD8
chimeras were isolated by immunoprecipitation (23) with protein
G-agarose before SDS-polyacrylamide gel electrophoresis (12% gel)
(24).
RESULTS The p24 family of proteins currently comprises 8 members in
mammals and yeast and various orthologues and homologues in other species (Fig. 1A) (10-16).
The alignment of p24 TMDs and cytoplasmic tails showed that in addition
to one absolutely conserved phenylalanine in the cytoplasmic domain
(position 195 in chop24a, the p24 protein isolated from CHO cells
(13)), other amino acids in the TMD are conserved. First, a glutamine
residue is present in the TMDs adjacent to the TMD-cytoplasmic domain
border in all p24s (position 187 in chop24a). Second, a position
juxtaposed to the exoplasmic domain (position 176 in chop24a)
accommodates a glutamic acid or polar residue in most p24 proteins with
only some exceptions in yeast (Fig. 1A). In a helical wheel
presentation, the TMD glutamic acid and glutamine (but not the strictly
conserved phenylalanine in the cytoplasmic domain) line the same side
of the predicted To analyze the role of the conserved residues on p24 localization
and trafficking, CD8 chimeras were generated in which the CD8 TMD and
cytoplasmic domain were replaced with the corresponding wild-type and
mutant sequences of chop24a (Fig. 1B) (13). The CD8 protein
has been used previously to analyze cytoplasmic domain and TMD
trafficking signals (25-27). CD8-chop24a chimeras were localized by
immunofluorescence in transiently expressing COS7 cells (Fig.
2). When the glutamic acid residue alone
(EA, panel A), in combination with the conserved
glutamine (EA-QA, panel C) or phenylalanines
(EA-FFAA, panel E), or all three positions (EA-QA-FFAA, panel F) were replaced with alanine,
the hybrid proteins were predominantly detected at the cell surface and
in a juxtanuclear area, just as the wild-type chimera had been
localized (cf. the previous analysis (20) and panel
G). In contrast, when the glutamine residue alone (QA,
panel B) or in combination with the phenylalanines (QA-FFAA, panel D) was replaced with alanine, the
chimeras were localized to the nuclear envelope and tubular-reticular
structures, presumably the ER (Fig. 2). These results suggest that the
presence of a glutamic acid in the TMD of p24 proteins confers
localization to the early secretory pathway when the conserved TMD
glutamine residue is absent.
To further corroborate this finding we performed pulse-chase
experiments to measure the rate and extent of transport of the chimeras
from the ER through the Golgi complex (Fig.
3A). The time required to
receive O-glycans (attached to the CD8 exoplasmic domain)
that were processed to the mature (sialic acid-containing) form in the
medial- or trans-Golgi (28) was similar for the EA, EA-QA,
and EA-FFAA mutant chimeras and comparable to the wild-type form (Fig.
3B) (cf. Ref. 20). The QA and QA-FFAA mutant
hybrid proteins, however, were not significantly processed within the 2-h chase period (Fig. 3).
Charged residues in the TMD confer efficient degradation of monomeric
subunits of the T cell antigen receptor in the absence of
oligomerization (29-31). But as expected from the structural context
of the chop24a glutamic acid residue (i.e. TMD length and
positioning) (31, 32), mutant CD8-chop24a chimera retained in the ER
were not rapidly degraded since more than 70% of the starting material
was present after a 2-h chase for all hybrid proteins.
Unexpectedly, the EA-QA-FFAA chimera was processed to the mature
form at a substantially higher rate than all other chimeras, including
the wild-type CD8-chop24a form (Fig. 3B) (cf.
Ref. 20). Apparently, the replacement of all three positions allowed
efficient transit from the ER through the Golgi complex.
Next, we tested whether the chop24a TMD alone alters the trafficking of
the reporter molecule CD8 by replacing the CD8 TMD with the respective
sequence of chop24a (data not shown). Pulse-chase experiments showed
that the rate of transport of the CD8-chop24a chimera was decreased
relative to the CD8 wild-type protein.
DISCUSSION Sorting signals in the TMD of mammalian and yeast proteins
are important for their localization to the Golgi complex and the ER
(27, 33-45), as well as for endocytosis and apical delivery in
polarized epithelial cells (46, 47). Recycling of the KDEL receptor and
Sec12p from the Golgi to the ER and retention of the coronavirus E1
protein in the Golgi are likely to require polar residues in the TMD of
the proteins (36, 38, 42, 48). For some proteins, trafficking
determinants have been found in the TMD as well as in the cytoplasmic
domain ( Unexpectedly, a EA-QA-FFAA CD8-chop24a hybrid protein that is
devoid of all analyzed sorting determinants was delivered to the
medial-/trans-Golgi at a significantly higher rate than the wild-type chimera and other hybrid proteins (Fig. 3). Apparently, the
presence of phenylalanine residues in the cytoplasmic domain decreases
the rate of transit from the ER through the Golgi complex when the TMD
glutamic acid and glutamine are absent. Conversely, when the TMD
glutamic acid and glutamine are present, the phenylalanine residues
increase the rate of transport (20). Moreover, the presence of the TMD
glutamine residue decreases the rate of transport when the TMD glutamic
acid and the phenylalanines are replaced with alanine. Taken together,
these observations suggest that the signals present in the TMD and
cytoplasmic domain modulate each other. The TMD determinant appears to
be transplantable since the exchange of the CD8 TMD alone with the
chop24a TMD significantly decreases the rate of transport from the ER
through the Golgi complex (data not shown).
For a variety of proteins the role of charged amino acids in the TMD
has been analyzed in detail. In subunits of the T cell antigen receptor
they are involved in quality control of hetero-oligomer assembly by the
formation of intramembrane charge pairs (29, 49) and in promoting the
formation of intramembrane disulfide-linked dimers (50). Since none of
the currently known p24 members contains basic residues or cysteines in
the TMD, similar mechanisms for p24 oligomerization seem unlikely.
Nevertheless, the recent analysis of the yeast members Emp24p and
Erv25p demonstrated that they can be efficiently cross-linked to each
other (15) suggesting that p24 proteins may form hetero-oligomers. It
has not been investigated whether p24 proteins can also form
homo-oligomers.
Several models could account for the differential localization and
trafficking of the CD8-chop24a chimeras. It has recently been shown
that similarly charged (glutamic acid) or polar (glutamine) TMD
residues can promote homodimerization and activation of the receptor
tyrosine kinase p185neu (51). In the context of p24 proteins
this could imply that the TMD is involved in oligomerization. If
oligomerization of p24 proteins (and CD8-chop24a chimeras) is required
for transport from the ER through the Golgi complex, then the TMD alone
is apparently not sufficient for rapid transit. The FFAA chimera in
which the phenylalanines in the cytoplasmic domain were replaced with
alanine (Fig. 3 (20)) was transported from the ER through the Golgi complex at a significantly lower rate than the wild-type form. Thus,
rapid transit requires the phenylalanine residues to aid oligomerization and/or to facilitate interaction with coatomer (20).
Although this model could explain the modulatory interactions among TMD
and cytoplasmic tail signals it does not account for the highly
efficient transport of the EA-QA-FFAA CD8-chop24a chimera. Moreover,
this premise does not provide an explanation for the apparent decreased
rate of transit of CD8-chop24a chimeras that retain only one of the
three positions, i.e. the TMD glutamine residue (EA-FFAA),
the glutamic acid (QA-FFAA), or the phenylalanine residues in the
cytoplasmic domain (EA-QA).
Precedence for an alternative model is provided from the analysis of
Sec12p in yeast. Two glutamines in the Sec12p TMD were shown to be
required for the recycling of escaped protein from the Golgi complex
back to the ER (42). Proper Sec12p ER localization required Rer1p, a
protein primarily localized to the Golgi complex (40-42, 52). Rer1p
contains four TMDs that encode for polar as well as charged residues
and is proposed to function as a TMD sorting receptor. A direct
interaction with Sec12p remains to be shown.
Analogous to Sec12p and Rer1p, p24 recruitment could involve not only
the interaction with coatomer (20) but also the binding to other
integral membrane proteins of the vesicle budding machinery. These
additional protein-protein interactions could ensure the availability
of essential machinery components at the budding site (e.g.
v-SNAREs (53)) and might be mediated by the TMDs of the interacting
proteins. Upon interaction with coatomer the p24 proteins would be
released into the budding vesicle. Alternatively, efficient recruitment
might require the simultaneous interaction of coatomer with both
factors which could either trigger dissociation of p24 proteins for
recruitment or lead to the recruitment of both components into the
budding vesicle.
With respect to our analysis of the CD8-chop24a chimeras the
second model predicts that the absence of any trafficking signal in the
TMD and a coatomer binding motif (e.g. phenylalanines or basic residues) would allow unregulated, passive inclusion into vesicles not restricted by the limited number of coatomer binding sites. This could explain the more rapid transport of the EA-QA-FFAA versus the EA-QA form of the CD8-chop24a chimeras.
Similarly, the absence of a TMD signal in a p24 protein (EA-QA-FFAA
chimera versus FFAA chimera) could abolish the interaction
with a negative regulatory component and effectively increase the rate
of transport by bulk flow.
It is possible that hitherto unidentified factors, or members of a
growing class of multispanning membrane proteins (54-60), are involved
in the regulation of p24 packaging into budding vesicles.
FOOTNOTES ACKNOWLEDGEMENTS We thank M. Veit, C. Hughes, and F. Parlati for critical comments on the manuscript and for discussions. We
are grateful to S. Ponnambalam and T. Nilsson for the CD8 cDNA and
to M. Spiess for the pECE vector.
REFERENCES
Volume 272, Number 40,
Issue of October 3, 1997
pp. 24739-24742
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
and
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Fig. 1.
The p24 proteins and CD8-chop24a
chimeras. A, the alignment of the COOH-terminal residues of
p24 proteins was generated with the GCG program pileup (61) using the
available sequences of 16 previously described p24 members (13, 20),
the T1/ST2-binding protein (10), and the novel entry hp24g and yp24h.
The expressed sequence tags coding for hp24g and yp24h were retrieved
by searching the NCBI dbest data base with the program Powerblast using
tblastn (available from ftp://ncbi.nlm.nih.gov/pub/sim2/PowerBlast). The prefix a refers to Arabidopsis thaliana,
h to human proteins, y to S. cerevisiae, chop24a is from CHO cells (13), gp25l is from dog
(11), p23 is from rabbit (12), T1/ST2BP is from human, and Emp24p (14)
and Erv25p (15) are from S. cerevisiae. The amino acid
sequences of the TMD and cytoplasmic domain are indicated in
single-letter code. The conserved phenylalanine/hydrophobic residues in
the cytoplasmic domain, the conserved glutamine, and the frequently
charged/polar residue in the TMD are boxed. GenBank/EMBL accession numbers are as follows. ap24a, Z34726; ap24b, T46519; chop24a, U26264; gp25l, X53592; p23, X98303; hp24a, X92098 (16); hp24b,
T48838, R25915, T17481; hp24d, T98284; hp24e, F07445; hp24g, assembled
from AA215037 and AA138141; Emp24p, X67317; yp24b, L22015; yp24c,
U00059; yp24d, L22015; yp24e, X87331 and T36996; yp24f, Z48432; Erv25p, Z49810; yp24h, Z72524. B, summary of CD8-chop24a chimeras. CD8 chimeras were constructed such that the 165 amino acids of the
human CD8 extracellular domain were preserved, followed by the amino
acids glycine-proline and the 34 residues of the chop24a TMD and
cytoplasmic domain. The wild-type chimera (wt) (20) and
mutant chimeras are shown. The glutamic acid (position 176 in chop24a),
glutamine (position 187 in chop24a), and phenylalanines (positions 194 and 195 in chop24a) that were replaced with alanine in the mutant
chimeras are indicated bold underlined.
[View Larger Version of this Image (77K GIF file)]
-helical structure (data not shown), revealing a
relatively nonhydrophobic face in the TMD of p24 proteins.
Fig. 2.
Intracellular localization of CD8-chop24a
chimeras by immunofluorescence microscopy. The chimeras were
analyzed in transiently transfected COS7 cells 46 h after
transfection. The hybrid proteins were expressed as EA (A),
QA (B), EA-QA (C), QA-FFAA (D),
EA-FFAA (E), EA-QA-FFAA (F), and wild-type forms
(G) (see Fig. 1B). The localization of the
wild-type and FFAA chimera have been described previously (20).
Untransfected cells are shown as a control in H. The cells
were incubated in medium containing 10 µg/ml cycloheximide for 2 h prior to fixation. They were permeabilized with Triton X-100 before
labeling with an antibody to CD8 and a secondary antibody. A set of
representative cells are shown. The exposure time and printing were
identical for A-H. Bar, 15 µm.
[View Larger Version of this Image (72K GIF file)]
Fig. 3.
Pulse-chase analysis of CD8-chop24a
chimeras. A, the mutant chimeras were analyzed in
transiently transfected COS7 cells 46 h after transfection. The
cells were labeled for 20 min with
[35S]methionine/cysteine and then incubated for 0, 15, 30, 45, 60, and 120 min in medium containing unlabeled
methionine/cysteine. Then, cells were lysed in Triton X-100, and the
CD8 chimeras were isolated by immunoprecipitation and analyzed by
SDS-PAGE. The CD8-chop24a precursor (p) and mature
(m) O-glycosylated forms (28) are indicated.
Immunoprecipitation from nontransfected cells is shown as a control
(Cont.). The wild-type and FFAACD8-chop24a chimeras have
been analyzed previously (20). B, quantitation of the
pulse-chase analysis. The amounts of the mature
O-glycosylated form (28) of each chimera were determined by
densitometric scanning of autoradiograms. The data points for the
EA-QA-FFAA and EA-QA CD8-chop24a chimeras represent average values from
two experiments, for the other hybrid proteins the analysis was carried
out once. The CD8-chop24a wild-type and FFAA mutant chimeras (20) are included for comparison.
[View Larger Version of this Image (29K GIF file)]
2,6-sialyltransferase, Sed5p, Sec12p, and TGN38) (27, 34,
37, 42). The chop24a member of the p24 family of putative cargo
receptors (13) encodes for a glutamic acid in the TMD. This residue
appears to serve as an ER retention signal which is attenuated by a
conserved glutamine in the TMD and phenylalanine residues in the
cytoplasmic tail (Fig. 2). The presence of all three motifs facilitates
efficient ER exit and transit through the Golgi complex.
To whom correspondence should be addressed: Cellular Biochemistry
and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-8445; Fax:
212-717-3604.
1
The abbreviations used are: ER, endoplasmic
reticulum; CHO, Chinese hamster ovary; TMD, transmembrane domain;
v-SNARE, vesicle-soluble (N-ethylmaleimide sensitive fusion
protein) attachment protein receptor; COP, coat protein.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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