(Received for publication, September 25, 1995; and in revised form, December 12, 1995)
From the
Vesicle traffic propagates and maintains distinct subcellular compartments and routes secretory products from their site of synthesis to their final destinations. As a basis for the specificity of vesicular transport reactions, each step in the secretory pathway appears to be handled by a distinct set of evolutionarily conserved proteins. Mammalian proteins responsible for vesicle trafficking at early steps in the secretory pathway are not well understood. In this report, we describe rat sec22 (rsec22) and rat bet1 (rbet1), mammalian sequence homologs of yeast proteins identified as mediators of endoplasmic reticulum-to-Golgi protein transport. rsec22 and rbet1 were expressed widely in mammalian tissues, as anticipated for proteins involved in fundamental membrane trafficking reactions. Recombinant rsec22 and rbet1 proteins behaved as integral membrane components of 28 and 18 kDa, respectively, consistent with their primary structures, which contain a predicted transmembrane domain at or near the carboxyl terminus. Recombinant rsec22 and rbet1 had distinct subcellular localizations, with rsec22 residing on endoplasmic reticulum membranes and rbet1 found on Golgi membranes. Studies with brefeldin A and nocodazole indicated that rbet1 function might involve interaction with or retention in the intermediate compartment. The distinct localizations of rsec22 and rbet1 may reflect their participation in opposite directions of membrane flow between the endoplasmic reticulum and Golgi apparatus.
Vectorial membrane transport reactions are presumed to require formation of protein complexes between integral components of the transport vesicle (vesicle SNAP receptors or v-SNARES) and target membrane (target membrane SNAP receptors or t-SNARES) as well as several soluble and peripheral membrane proteins. Because distinct sets of these proteins are stationed at each membrane transport step, it has been proposed that they encode the specificity and fidelity of membrane trafficking reactions(1, 2) . Despite characterization of several protein complexes involving integral proteins of the vesicle and target membranes, homologs of the yeast Sec1 protein, and rab proteins, it is not known how protein-protein interactions contribute to the specificity of targeting, docking, and fusion of transport vesicles with the target membrane.
Mechanistic insights from higher eukaryotes have been gained through the molecular characterization of nerve terminal components (3) and the study of in vitro membrane transport reconstitutions(4) . Although protein-protein interactions sufficient to explain aspects of docking and fusion have been documented in the nerve terminal, where components are abundant(5, 6) , the anticipated array of proteins involved at distinct transport steps has not appeared. Therefore, opportunities to evaluate the role of various protein complexes in the specificity of docking and fusion depend upon the discovery of additional vesicle trafficking proteins from distinct cellular compartments.
BET1, BOS1, and SEC22 form
a set of interacting yeast genes required in endoplasmic reticulum
(ER)()-to-Golgi protein transport. Overexpression of BOS1 suppresses bet1 mutations, and overexpression of
either BET1 or BOS1 suppresses sec22 mutations(7, 8) . The accumulation of vesicles
seen in sec22 mutants indicates that this mutation affects a
late, post-budding step, perhaps vesicle targeting or fusion (9) . Each of these genes encode small integral membrane
proteins (16, 27, and 28 kDa, respectively) with hydrophilic
cytoplasmically oriented N termini and central domains and C-terminal
transmembrane anchors. Their small size and overall membrane topology
are reminiscent of the vesicle-associated membrane protein (VAMP)
family(10) . Additionally, Bet1p, Bos1p, and Sec22p have all
been detected as integral components of putative ER-to-Golgi transport
vesicles, although it is controversial as to whether all three reside
on a single vesicle class (11, 12) . There appears to
be a consensus at least that Bos1p and Sec22p exist on a single vesicle
type, and there is recent evidence that they physically interact to
activate vesicles for docking or fusion with the Golgi
membrane(13) . The transport vesicles also appear to contain
the GTP-binding protein Ypt1p(11) , whose function in
ER-to-Golgi transport has been suggested to involve regulation of the
Bos1p-Sec22p interactions(13) .
Yeast Sed5p (14) and its mammalian homolog syntaxin 5 (15) represent potential receptors on the Golgi membrane for targeting proteins of ER-derived transport vesicles. Recently, large protein complexes containing Sed5p, Bet1p, Bos1p, Sec22p, Sly1p, and Sec17p as well as previously uncharacterized proteins have been immunoprecipitated from detergent extracts of yeast cells arrested at a late step in ER-to-Golgi protein transport(16) . Although consistent with the expectation that vesicle targeting and fusion require protein complex formation between transport step-specific proteins, these findings highlighted the need to define specific roles for the unexpectedly large number of protein participants.
To further understand the mechanism of vesicle transport, we sought mammalian homologs of Sec22p and Bet1p. The presence of mammalian sequence homologs to proteins that mediate ER-to-Golgi traffic in yeast confirms the universal nature of the machinery underlying steps in the secretory pathway. As a first indication of these proteins' function, we expressed and localized them in mammalian cells with distinctive and well characterized morphology. Unexpectedly, the two proteins resided in membranes at opposite ends of the anticipated transport step, with rsec22 on ER membranes and rbet1 on Golgi membranes. One suggestion is that rsec22 and rbet1 are involved in the trafficking of vesicles participating in opposite directions of transport between the ER and Golgi.
The human bet1 PCR probe was utilized to screen a total of
3 10
plaques from a Stratagene
ZAPII adult rat
brain cDNA library, resulting in a single positive clone, B-bet1-1,
harboring a 1.4-kb cDNA. Sequencing revealed that this clone encoded
amino acids 37-118 of a rat bet1 sequence homolog (rbet1), but
was truncated at the 5`-end, as was the human EST. A 1.2-kb restriction
fragment from the incomplete clone was labeled and employed to screen a
total of 5
10
plaques from a brain cDNA library,
1.075
10
from pancreas, and 1.25
10
from liver. Several more truncated clones were discovered in both
the pancreas and liver libraries, as well as a single liver clone,
designated LS2, that appeared to be full-length. LS2 contained an open
reading frame of 407 nucleotides within a cDNA insert of 1.3 kb.
Sequencing of both strands of the open reading frame revealed that the
methionine representing the first amino acid of rbet1 occurred 54 bp
downstream of a stop codon and would be predicted to efficiently
initiate translation(17) . Since several truncated clones had
been discovered and since rbet1 appeared to be 24 amino acids shorter
at the amino terminus than yeast Bet1p, we confirmed that LS2 encoded
an authentic amino terminus by PCR cloning the open reading frame from
an independent (skeletal muscle) cDNA library and found it had an
identical rbet1 amino terminus. GenBank and EMBL data base searches
revealed that rbet1 was most similar to two ESTs (accession numbers
T31927 and Z45699) that appeared to encode portions of a human Bet1p
homolog 87% identical to rbet1. A rice EST (accession number D23851)
encodes a predicted protein 32% identical to rbet1 and 17% identical to
yeast Bet1p.
Sequence alignments were performed using the BESTFIT program (Genetics Computer Group) using default parameters (gap weight = 3 and gap length weight = 0.10) and the option that provides quality scores from 100 additional alignments using randomized sequences. The alignments presented in Fig. 1and Fig. 2were generated as described above using the full-length sequences; then sequence ends truncated by the program were restored, and all markings except identities and pairs of dots between conservative similar pairs (see Fig. 1legend) were removed. Percent identity was 32% (rsec22 and Sec22) and 21% (rbet1 and Bet1). The z number was 12.9 for the rsec22 alignment and 9.5 for the rbet1 alignment. z numbers were calculated from the quality scores provided by the BESTFIT program: z = (quality score of alignment in question - mean quality score of 100 alignments using randomized sequences)/S.D. of 100 quality scores from randomized sequences.
Figure 1: Comparison of deduced amino acid sequences of rsec22 and yeast Sec22p (ysec22). Predicted transmembrane sequences are underlined. The two proteins display 32% identity over a 202-amino acid overlap. Vertical bars separate pairs of identical residues; two dots designate similar pairs of amino acids with the following groupings considered similar: R and K; Q and N; T and S; E and D; and V, I, L, F, and M.
Figure 2: Comparison of deduced amino acid sequences of rbet1 and yeast Bet1p (ybet1). Predicted transmembrane sequences are underlined. The two proteins display 21% identity over a 116-amino acid overlap. Identical and similar residues are designated as described for Fig. 1.
COS cells (passages 3-15) were maintained in
Dulbecco's modified Eagle's medium H-16 with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin
sulfate in a humidified 5% CO incubator. Cells (5
10
) were plated on 10-cm dishes
24 h prior to
transfection. For transfections, plates were rinsed twice with
Dulbecco's modified Eagle's medium H-16 lacking serum and
antibiotics and incubated for 40 min at 37 °C with 5 ml of the same
medium containing 2.5 mg of DEAE-dextran and 25 µg of pCMV
construct DNA purified with QIAGEN maxi- or midiprep kits. Plates were
then incubated at 37 °C with 5 ml of Dulbecco's modified
Eagle's medium H-16, 10% fetal bovine serum, and antibiotics plus
100 µM chloroquine. After 2 h, the plates were incubated
for 2.5 min at room temperature in 5 ml of Dulbecco's modified
Eagle's medium H-16, fetal bovine serum, and antibiotics plus 10%
dimethyl sulfoxide and then returned to 37 °C in normal culture
medium. The following day, culture medium was replaced, and/or the
cells were replated on microscope slides. Transfected cells were fixed
for microscopy or harvested for extraction studies 40-48 h
post-transfection.
Cell staining
involved a 15-min incubation in permeabilization solution (0.4%
saponin, 1% bovine serum albumin, and 2% normal goat serum in PBS)
followed by incubation in permeabilization solution containing primary
antibody for 1 h at room temperature. Cells were then washed three
times with PBS and incubated in permeabilization solution containing
affinity-purified fluorescein- or rhodamine-labeled goat antibodies
against mouse or rabbit IgG (BioSource, International, Camarillo, CA).
Slides were finally washed three times with PBS, covered with Citifluor
antiquenching agent (Citifluor, Canterbury, United Kingdom), and
mounted beneath coverslips. Microscopy and photography were conducted
using a Zeiss Axiophot microscope/camera system using a 40 or
63
oil objective for a total magnification of
125 or
197 and Ektachrome 400 ASA film. Color slides were digitized
and then cropped, adjusted, and arranged using Adobe Photoshop software
and printed using a photodigital printer.
Rabbit syntaxin 5 antiserum was raised by injection of a bacterial glutathione S-transferase fusion protein including the syntaxin 5 cytoplasmic domain and was useful for immunostaining at a dilution of 1:1000. Four observations demonstrated that the Golgi staining obtained with this antiserum was specific for syntaxin 5. First, the localization matched that seen previously with epitope-tagged syntaxin 5(15) . Second, the antiserum recognizes recombinant syntaxin 5 overexpressed in COS cells, as indicated by a dramatic increase in Golgi staining intensity relative to surrounding nontransfected cells (data not shown). Third, preimmune serum did not exhibit staining in COS cells (data not shown). Fourth, an excess of glutathione S-transferase-syntaxin 5 bacterial fusion protein eliminated the Golgi staining in COS cells, whereas glutathione S-transferase alone had no effect (data not shown).
Figure 6:
Epitope-tagged rsec22 (A) and
rbet1 (B) behave as integral membrane proteins in transfected
COS cells. Post-nuclear membrane pellet fractions were extracted with
various disruptive agents and centrifuged at 100,000 g, and the resulting supernatant (S) and pellet (P) were analyzed by SDS-polyacrylamide gel electrophoresis
and immunoblotting with anti-myc monoclonal antibodies. The
membranes in the first and second lanes were
extracted with 1.5 M NaCl at pH 7.0. For the third through eighth lanes, membranes were first extracted with
1.5 M NaCl and then with 5 M urea at pH 7.0 (third and fourth lanes), 0.2 M sodium
carbonate at pH 11.4 (fifth and sixth lanes), or 1%
Triton X-100 at pH 7.0 (seventh and eighth lanes). myc-rsec22 migrated as a 28-kDa protein, and myc-rbet1 as an 18-kDa protein. myc-rsec22 and myc-rbet1 could be detected only in membrane fractions and
only in transfected COS cells (not shown).
The rsec22 open reading frame predicts a 28.5-kDa integral membrane protein with hydrophilic termini and central regions and a transmembrane domain positioned 46 amino acids from the C terminus. No signal sequence was evident. rsec22 displayed 32% identity to yeast Sec22p when optimally aligned (Fig. 1). The identities were distributed approximately evenly over the sec22 proteins and not confined to one region of the molecule. The rsec22 putative transmembrane domain (amino acids 187-208) corresponded to the yeast transmembrane domain (amino acids 189-213). A region containing both hydrophobic and hydrophilic residues extended 46 amino acids beyond the putative transmembrane domain, perhaps representing a luminal domain. The VAMP synaptic vesicle proteins from Drosophila and Aplysia possess luminal domains of up to 80 amino acids of unknown function, whereas yeast Sec22p and rat VAMP do not. On the other hand, the rsec22 C-terminal region could instead encode a second transmembrane domain since amino acids 225-247 contain 13 hydrophobic and no charged residues. A second transmembrane domain would position the charged C-terminal seven residues in the cytoplasm rather than the lumen.
The rbet1 open reading frame predicts an integral membrane protein of 13.2 kDa. rbet1 possesses a truncated amino terminus relative to yeast Bet1p and displays 21% amino acid identity (Fig. 2). The optimal alignment quality score of rbet1 with Bet1p was 9.5 standard deviations above the mean score obtained using randomized sequences (z = 9.5; see ``Experimental Procedures''). Amino acid identities were most abundant around amino acid 48, perhaps highlighting a region of conserved function from yeast to mammals. rbet1 also displays a putative transmembrane domain (amino acids 96-115) corresponding to the yeast hydrophobic stretch (amino acids 118-141), but unlike rsec22, lacks a significant C-terminal extension beyond the predicted transmembrane sequence. As with rsec22, no signal sequence was evident.
GenBank and EMBL data base searches revealed that rsec22 was more similar to yeast Sec22 than to any other protein or predicted protein sequence. rbet1 was most similar to two human ESTs (see ``Experimental Procedures'') that appeared to encode portions of a human version of rbet1. A rice EST sharing 32% identity with rbet1 and 17% identity with yeast Bet1p also recently appeared in the data base.
Despite clear relatedness of rsec22 and rbet1 to their yeast counterparts, neither is particularly similar in sequence to other mammalian vesicle trafficking proteins such as VAMP and cellubrevin, although their overall structure, size, and membrane topology are characteristic. Pairwise optimal alignments revealed that none of the stretches of three or more residues identical between rsec22 and Sec22 or rbet1 and Bet1 (see Fig. 1and Fig. 2) were conserved between any one of these four proteins and rat or bovine VAMP/synaptobrevin isoforms (data not shown). The patterns of conservation do not readily distinguish amino acid stretches likely to be involved in general versus transport step-specific functions.
One feature of VAMP and related proteins that may be important in protein complex formation is the predicted existence of coiled-coil structures in the cytoplasmic domains. It has been suggested that coiled-coil interactions form the basis of interactions between vesicle and target membrane proteins, although no definitive structural data have been presented(25) . We analyzed the rsec22, Sec22, rbet1, and Bet1 sequences for structures likely to engage in coiled-coil interactions using the COILS program (26) and found that yeast Sec22 is the only protein among them with a substantial probability to contain a coiled-coil domain (rsec22, 0.03% probability of forming a coiled-coil domain; Sec22, 60%; rbet1, 16%; and Bet1, 8.5%). These predictions contrast with that for rat VAMP-2, which exhibits a 95% probability of forming a coiled-coil domain. Hence, if rsec22 and rbet1 participate in the protein complexes proposed to control vesicle transport steps, their participation may involve interaction mechanisms other than coiled coil-based binding. The significance and universality of coiled-coil mechanisms in vesicle trafficking are major issues yet to be resolved.
Figure 3: Multiple-tissue Northern blot using rsec22 (A) and rbet1 (B) cDNA probes. The major mRNA species recognized with the rsec22 probe has a predicted size of 2.6 kb; the major species for rbet1 is 1.7 kb. The autoradiogram exposure shown for rsec22 was 4 h, while that for rbet1 was overnight. Upon overnight exposure, the rsec22 mRNA was visible in all of the lanes (not shown).
Figure 4:
Double-label immunofluorescence microscopy
showing the intracellular localization of epitope-tagged rsec22 and
rbet1 in transfected COS cells. A and C, myc-rsec22 stained with anti-myc monoclonal
antibodies; B and D, endoplasmic reticulum stained
with anti-calnexin antiserum; E, myc-rbet1 stained
with anti-myc monoclonal antibodies; F, cis-Golgi membranes stained with -COP antiserum. Bar = 25 µm (A, B, E, and F) and 17 µm (C and D).
Figure 5: Double-label immunofluorescence microscopy documenting the dynamics of epitope-tagged rbet1 and endogenous syntaxin 5 upon BFA and/or nocodazole treatment of transfected COS cells. 2-h treatment with 10 µM nocodazole: A, myc-rbet1 stained with anti-myc monoclonal antibodies; B, syntaxin 5 stained with anti-syntaxin 5 antiserum. 2-h treatment with 10 µg/ml BFA: C and E, myc-rbet1; D, syntaxin 5; F, p58, an intermediate compartment protein, stained with anti-p58 antiserum. 2-h treatment with 10 µg/ml BFA followed by 1-h treatment with 10 µg/ml BFA plus 10 µM nocodazole: G, myc-rbet1; H, syntaxin 5. 1-h treatment with 10 µg/ml BFA followed by 4 h of recovery in normal growth medium: I, myc-rbet1; J, syntaxin 5. Bar = 15 µm (A-F, I, and J) and 34 µm (G and H).
BFA causes a block in ER-to-Golgi membrane traffic and induces Golgi proteins to return to the ER via a retrograde tubulovesicular mechanism. Since rsec22 may participate dynamically in ER-to-Golgi membrane flow, we tested whether a brefeldin A blockade of ER-to-Golgi traffic would produce an altered myc-rsec22 distribution. We found that myc-rsec22 and the ER resident protein calnexin continued to colocalize after 2 h of treatment with 10 µg/ml BFA, indicating that epitope-tagged rsec22 was not dependent on normal ER-to-Golgi traffic to maintain its characteristic ER localization (data not shown). We also examined the behavior of the Golgi vesicle trafficking proteins syntaxin 5 and myc-rbet1 as well as the 58-kDa Golgi protein upon BFA treatment. The 58-kDa protein has been previously shown to redistribute to the ER under similar conditions (34) . Syntaxin 5 (Fig. 5D), like the 58-kDa protein (data not shown), took on a fine reticular pattern spreading throughout the cytoplasm and often encompassing the nuclear envelope. This fine reticular pattern is consistent with the behavior of Golgi resident proteins that redistribute to the ER(30, 32) . We also observed spotty vesicular staining for syntaxin 5 (Fig. 5D) and, to a lesser extent, the 58-kDa protein (data not shown), although in most cases, this did not appear markedly concentrated relative to the continuous reticular pattern. myc-rbet1 also exhibited a fine reticular pattern upon BFA treatment, indicating access to the ER; on the other hand, myc-rbet1 more often displayed a marked spotty appearance, with bright staining of cytoplasmic vesicles, fainter reticular staining, and little or no visible coverage of the nuclear envelope (Fig. 5, C and E). In BFA-treated transfected cells costained for myc-rbet1 and syntaxin 5, the spotty structures decorated by both antibodies coincided, the difference being that myc-rbet1 appeared more concentrated in the vesicular structures, whereas syntaxin 5 appeared more diluted into the fine reticular pattern encompassing the cytoplasm and often covering the nuclear envelope (Fig. 5, C versus D). The greater relative concentration of myc-rbet1 in cytoplasmic vesicles was not a transient phase during BFA treatment since it was manifest after 1, 2, 3, and 4 h of 10 µg/ml BFA treatment. It was not a distinction caused by overexpression since many cells exhibiting very weak myc-rbet1 staining levels also displayed predominantly spotty staining. We also found that endogenous syntaxin 5 and hemagglutinin-tagged syntaxin 5 (15) produced similar, mostly reticular staining patterns upon BFA treatment, whether detected with syntaxin 5 antiserum or anti-hemagglutinin monoclonal antibodies (data not shown).
Although Golgi resident proteins are primarily visible in the ER during BFA treatment, they have been shown to rapidly cycle between the ER and the intermediate compartment via a microtubule-independent anterograde pathway and a microtubule-dependent retrograde shunt mechanism(32) . To determine if the spotty structures visible during BFA treatment represented intermediate compartment vesicles, we costained myc-rbet1-transfected cells for myc-rbet1 and p58, a protein that persists in vesicular intermediate compartment elements during BFA treatment(35) . As seen in Fig. 5(E and F), the vesicles containing myc-rbet1 extensively overlap with the structures containing p58, suggesting that myc-rbet1 has access to the intermediate compartment during BFA treatment. To determine if myc-rbet1 and syntaxin 5 cycle between the ER and the intermediate compartment in the presence of BFA, we treated cells for 2 h with BFA followed by 1 h with BFA plus 10 µM nocodazole to block retrograde movement to the ER. This treatment causes Golgi proteins participating in BFA-induced ER-intermediate compartment cycling to accumulate in the intermediate compartment(32) . As shown in Fig. 5(G and H), this treatment markedly intensified the coincident spotty vesicular staining of myc-rbet1 and syntaxin 5, indicating that both proteins visited intermediate compartment vesicles transiently during BFA treatment. We found a similar intensification of colocalizing spotty staining for the 58-kDa protein as well (data not shown).
Our results indicate that the Golgi vesicle trafficking proteins myc-rbet1 and syntaxin 5 follow the pathway taken by the 58-kDa Golgi protein and several other Golgi resident proteins (30) during BFA treatment. This pathway includes access to the ER as well as to cytoplasmic intermediate compartment vesicles. This is in contrast to proteins that persist stably in the intermediate compartment(31, 35) , fuse with endosomal compartments(36, 37) , or become cytosolic (34) in response to BFA. The apparent tendency of myc-rbet1 to concentrate in intermediate compartment vesicles during BFA treatment could indicate participation in an intermediate compartment retention mechanism. Perhaps normal rbet1 function includes interaction with this compartment. On the other hand, a slower depletion from the Golgi membrane in response to BFA, via intermediate compartment vesicles, could account for the marked staining of cytoplasmic vesicles. Such a sluggish retrograde movement from the Golgi could indicate participation in Golgi retention mechanisms.
The kinetics of BFA effects on myc-rbet1 and syntaxin 5
differed. Whereas the tight perinuclear syntaxin 5 staining was well
dispersed within 5 min of BFA addition, the tight perinuclear myc-rbet1 staining was often not well dispersed until 1 h
after BFA addition. BFA disruption of both proteins was fully
reversible, with return of a compact Golgi localization over 2-7
h following a 1-h BFA exposure (Fig. 5, I and J). Despite the different rates observed for initial
disruption and the difference in the concentration in intermediate
compartment elements, myc-rbet1 and syntaxin 5 staining
returned in parallel to their perinuclear location following removal of
BFA. In no case did syntaxin 5 or myc-rbet1 appear to
reassemble into a compact Golgi structure while the other protein
remained in dispersed elements. Hence, different mechanisms limit the
rate at which these proteins initially traverse the retrograde pathway
out of the Golgi membrane and finally return via anterograde movement
to the newly reformed Golgi membrane. We observed that the reassembly
of compact perinuclear staining was retarded in myc-rbet1-transfected cells relative to nontransfected COS
cells or myc-rsec22- or myc-rbet1TM-transfected
(see below) cells (data not shown). After 4 h of recovery from BFA, a
compact perinuclear syntaxin 5 staining was evident in 22% of cells
transfected with myc-rbet1, 42% of cells transfected with myc-rsec22 or myc-rbet1
TM, and 37% of
nontransfected COS cells. By 7 h post-BFA treatment,
47% of cells
exhibited a compact perinuclear distribution of syntaxin 5, regardless
of their transfection status. 50% is approximately the fraction
exhibiting this morphology in untreated cells, the remainder being
aberrations due to multinucleated or otherwise misshapen cells or poor
staining. Although this effect of the myc-rbet1 protein is not
uniquely interpretable in terms of rbet1 function, it may indicate that
rbet1 can modulate the delicate balance of membrane flow into and out
of the Golgi, consistent with its potential role as a vesicle
trafficking protein for this dynamic organelle.
For both myc-rsec22 and myc-rbet1, only a single, transfection-specific immunoreactive protein band was observed (data not shown). myc-rsec22 had an SDS gel mobility of 28 kDa, closely matching its predicted molecular mass. The myc-rbet1 band appeared larger than predicted from its sequence (18 versus 13.2 kDa for rbet1 and 1.1 kDa for the myc tag), a feature shared with VAMP and which could indicate the presence of post-translational modifications or SDS-resistant secondary structures.
Yeast Sec22p has been observed to associate
tightly with another potential vesicle targeting protein, Bos1p, on
ER-to-Golgi transport vesicles(13) . A propensity to undergo
protein-protein interactions could contribute to the localization of
these trafficking proteins at their proper membrane station. This
notion led us to express and localize by immunofluorescence in COS
cells myc-rsec22 and myc-rbet1 proteins with
C-terminal deletions removing the putative membrane anchors. myc-rsec22 lacking a putative transmembrane domain and luminal
sequence (designated myc-rsec22TM) appeared diffuse
throughout the cell and no longer visibly associated with ER tubules
(data not shown). This indicated a requirement for the rsec22
transmembrane and/or luminal domain for membrane attachment and
association with ER elements. myc-rbet1 lacking its putative
transmembrane sequence (myc-rbet1
TM) also exhibited a
diffuse staining pattern (data not shown), indicating that the 23-amino
acid putative transmembrane sequence was required for both membrane
association and Golgi localization of myc-rbet1. Since it was
possible that the deleted proteins could have dominant effects on the
secretory pathway in COS cells, we examined the morphology and staining
pattern of several secretory organelles in cells expressing myc-rsec22
TM and myc-rbet1
TM. We found no
visible change in ER membranes as marked by calnexin, in the
intermediate compartment as marked by p58, in the Golgi membrane as
marked by
-COP and syntaxin 5, in the trans-Golgi/trans-Golgi network as marked by wheat
germ agglutinin, in the trans-Golgi network/endosomes as
marked by the mannose phosphate receptor, or in the apparent
distribution of a constitutively secreted cargo protein,
peptidylglycine
-amidating monooxygenase (see ``Experimental
Procedures''), cotransfected with the epitope-tagged proteins.
These data suggest that the deleted constructs did not exert a dominant
effect on the secretory pathway, an outcome possibly explained by their
inability to interact with the proper membrane compartment. Note that
relatively small perturbations in vesicle trafficking might not have
been detectable using these morphological criteria.
Despite these similarities, we cannot rule out the possibility that rsec22 and/or rbet1 participates in steps other than ER-to-Golgi transport. rsec22 or rbet1 may represent a vesicle transport protein involved at distinct transport steps for which yeast proteins have not yet been recognized or correctly assigned. As an example, rbet1 could mediate intra-Golgi rather than ER-to-Golgi transport. Perhaps the present rbet1 is one of several Bet1-like proteins that mammalian cells have evolved for distinct transport steps. It is not yet known how many homologs of yeast Sec22p and Bet1p exist in mammalian species, or if the two proteins characterized here are the most similar homologs.
Although both rsec22 and rbet1 localized to compartments within the anticipated transport step, it is noteworthy that they were observed only on the end point compartments (ER and Golgi), rather than on transport vesicles or other intermediates. Since yeast Sec22p and Bet1p have been biochemically detected on putative ER-to-Golgi transport vesicles, this was somewhat surprising. This situation differs from that of the mammalian vesicle transport protein VAMP/synaptobrevin, where localization to vesicles is manifest(40) . Unlike synaptic vesicles, ER-to-Golgi transport vesicles are not anticipated to accumulate under normal conditions and hence might not be evident by fluorescence microscopy. Since vesicular transport between the ER and the Golgi membrane must be rapid and continuous, the total pool of each required vesicle protein should be substantially larger than that pool present on transport vesicles at any given time, with the resting pool accumulating on the donor, the acceptor, or both end compartments. It thus seems possible that rsec22 and/or rbet1 represents a transport vesicle component, as predicted based on their yeast sequence homologs, but are only evident at one end point of the transport cycle. A simple explanation for their opposing localizations would be that rsec22 and rbet1 may be involved in opposite directions of transport between ER and Golgi membranes. However, the possibility that rsec22 and/or rbet1 does not represent a transport vesicle component should also be considered since their localizations are also compatible with ER or Golgi resident trafficking roles.
The behavior of Golgi vesicle trafficking proteins in the presence of BFA had not been previously reported. We found that BFA induced myc-rbet1 to enter an ER-intermediate compartment cycling pathway like syntaxin 5 and most previously characterized Golgi proteins. However, myc-rbet1 responded more slowly to BFA and accumulated more profoundly in intermediate compartment vesicles. This behavior may indicate that rbet1 participates in Golgi and/or intermediate compartment retention mechanisms, possibly involving oligomerization, but does not uniquely favor a resident versus a vesicular function. The discovery of mammalian trafficking proteins that localize to the ER and Golgi apparatus provides valuable reagents for mechanistic studies that will undoubtedly entail in vitro transport assays(41) , providing functional correlates for protein-protein interactions involving a growing collection of trafficking proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U42209 [GenBank]and U42755[GenBank].