In the present study, we have demonstrated that cellubrevin specifically interacts with BAP31. BAP31 is a resident of the ER that probably shuttles between the ER and the intermediate compartment and/or cis-Golgi complex. Our data suggest that BAP31 binds newly synthesized cellubrevin, and perhaps other proteins, to control their export to the Golgi apparatus where these transported proteins reach their final destinations.
As a resident of the ER, BAP31 does not colocalize with the major pools of cellubrevin. In addition, we found BAP31-positive membranes concentrated in a paranuclear region close to the Golgi apparatus and the MTOC, an area that is devoid of lumenal ER proteins. Proteins involved in endosomal recycling, such as cellubrevin and the transferrin receptor, are also concentrated but, as our analysis indicates, they reside on different vesicle populations. This area apparently serves as a central relay station for trafficking vesicles of different origins and destinations.
Export from the ER, the first step in the vectorial transport of proteins, commences in specialized regions of the ER. In some cells, e.g., the pancreatic acinar cells, these regions are juxtaposed to the cis
-Golgi network and were originally referred to as transitional elements (Palade, 1975
). In other cells, they appear to be distributed throughout the cytoplasm (Bannykh et al., 1996
, Presley et al., 1997
). They represent regions of the ER with many budding vesicles that are often adjacent to vesiculo-tubular clusters (Saraste and Svensson, 1991
; Balch et al., 1994
). Budding from the ER involves COPII coat proteins and results in the formation of COPII-coated transport vesicles (Barlowe et al., 1994
). Before reaching the cis
-Golgi, these transport vesicles pass through vesiculo-tubular clusters that may represent the intermediate compartment, functionally defined as the sorting compartment between the ER and the Golgi complex (Aridor and Balch, 1996
). Here, ER resident proteins are probably sorted out and transported retrogradely to the ER, presumably involving COPI-coated transport vesicles (Aridor and Balch, 1996
; Bannykh et al., 1996
; Schekman and Orci, 1996
). The accumulation of BAP31-containing vesicles around the MTOC, an area devoid of lumenal ER proteins, demonstrates clearly that the protein exits the ER during its life cycle. However, it remains to be established whether it is transported all the way to the cis
-Golgi. Since we found only minor colocalization with the cis
-Golgi marker β-COP, its steady-state concentration in that compartment must be low and the time BAP31 resides in these cisternae very short. Additionally, the protein may be sorted out earlier and shipped back to the ER by retrograde transport. It should be emphasized, however, that the evidence that supports recycling of BAP31 from these compartments to the ER is indirect. Thus, we cannot exclude that BAP31 is directed to lysosomes where it is degraded instead of returning to the ER. We regard this as less likely because BAP31 does not exhibit a lysosomal staining pattern and it is completely absent from purified clathrin-coated vesicles (Fig. ).
Upon nocodazole treatment, the BAP31 staining in the region of the MTOC disappeared and BAP31 was almost exclusively retained in the ER with no overlap with cis
-Golgi markers (our unpublished observations). Thus, nocodazole blocks forward transport of BAP31-containing vesicles from the ER to the MTOC, in addition to its inhibition of retrograde transport that is known to be microtubule dependent (Lippincott-Schwartz et al., 1990
). If only retrograde transport was inhibited by the drug, BAP31 would be expected to accumulate in the cis
-Golgi area. Forward transport between the ER and the Golgi dependent on microtubules is in agreement with recent observations (Bannykh et al., 1996
; Rowe et al., 1996
; Presley et al., 1997
). Nocodazole also disrupted the accumulation of vesicles containing transferrin receptor and cellubrevin in this area (Daro et al., 1996
), making the differential distribution of BAP31 and cellubrevin more obvious. These findings highlight the role of the MTOC as a central relay station for microtubule-based intracellular vesicle traffic. Apparently, both ER-derived forward trafficking vesicles and plasmalemma- or endosome-derived endocytic vesicles are collected by microtubular transport from the cell periphery and then passage through the area of the MTOC before reaching their destinations at the cis
- and trans
-side of the Golgi complex, respectively.
It remains to be established which domains of BAP31 are responsible for its intracellular sorting. Deletion of the KKXX motif (which functions in the recruitment of COPI proteins), as well as deletion of a longer stretch (including the YDRL motif), had no obvious effects on the localization of the protein. Apparently, the KKXX motif is redundant to another as yet unknown sorting signal, and probably has a secondary signal function in assisting other proteins in the recruitment of COPI. It is possible, however, that mutant BAP31 is associated with endogenous wild-type BAP31 that still contains intact sorting signals. Interestingly, BAP31 remained in the ER even when the entire cytoplasmic tail was deleted, although upon extended culturing, abnormal vesicles were observed and cell viability decreased (see below).
Although cellubrevin and BAP31 are localized to different subcellular membranes, the interaction between these two proteins is highly specific. Like synaptophysin for synaptobrevin, BAP31 appears to be the dominant binding protein for cellubrevin, clearly exceeding the still elusive putative SNARE partners of the protein. Binding was observed with native as well as with recombinant proteins, suggesting that the interaction is direct and does not require intermediate proteins. Furthermore, binding appears to require the transmembrane domains of both BAP31 and cellubrevin, although electrostatic interactions must also be involved that can be shielded by high ion concentrations. Despite the specificity and affinity of the interaction, only a relatively small proportion of the proteins are complexed in cellular detergent extracts. This finding agrees well with the differential localization of the proteins and indicates they are associated with each other only during the early phase of the intracellular traffic of cellubrevin. Since cellubrevin, like synaptobrevin, is probably synthesized on free ribosomes (Kutay et al., 1995
), we assume that it interacts with BAP31 after posttranslational insertion into the ER membrane, although an additional role of BAP31 in membrane insertion of cellubrevin cannot be excluded at present.
How does BAP31 influence the export of cellubrevin from the ER? Two explanations are possible. First, BAP31 may function as a negative regulator that retains (or even recruits) newly synthesized cellubrevin in the ER until it is released by an unknown regulatory mechanism. Second, BAP31 may function as a positive regulator to which cellubrevin needs to bind to reach the Golgi compartment. According to this scenario, cellubrevin would be unable to leave the ER unless it is recruited by BAP31 into export vesicles, i.e., being actively transported rather than passively sorted by bulk flow.
According to the first view, BAP31 would bind to cellubrevin and other to be exported proteins and keep them in the ER membrane until they are either assembled with other membrane components or properly folded. However, we found that under all experimental conditions, only a fraction of cellubrevin is associated with BAP31 in detergent extracts. Neither treatment with nocodazole (resulting in a minor increase of cellubrevin in the ER) nor expression of a BAP31 mutant lacking the cytoplasmic domain (resulting in retention of cellubrevin in the ER), led to a noticeable increase of cellubrevin–myc
–BAP31-TMR complexes relative to the uncomplexed protein pools. Although artefacts can never be excluded when assessing membrane protein complexes in detergent extracts, association of the proteins appears to be low even if they are both confined to the ER. This finding is difficult to reconcile with the negative regulator model. Rather, it suggests that BAP31 may serve as a sorting chaperone that recruits certain classes of membrane proteins into transport vesicles. BAP31 may directly interact with COPII budding components. Alternatively, it may bind (perhaps via its coiled coil regions) to integral membrane proteins that facilitate COPII-mediated export. In fact, there are precedents for protein-assisted export from the ER in yeast. For instance, the protein Erp25p forms a complex with Emp24p that is required for selective export of certain cargo molecules from the ER (Schimmöller et al., 1995
; Belden and Barlowe, 1996
). Another well-documented case is the yeast gene product Shr3p that, similar to BAP31, resides primarily in the ER and recruits specific amino acid permeases into transport vesicles (Kuehn et al., 1996
Deletion of the cytoplasmic tail of BAP31 results in the retention of both BAP31 and cellubrevin in the ER, whereas other proteins such as the transferrin receptor still reach their normal destination beyond the Golgi complex. Moreover, the differences between transferrin receptor and cellubrevin localization are not caused by differences in the turnover rates of the proteins because preliminary observations suggest that their half lives are similar. Thus, these findings document that once the function of BAP31 is impaired, cellubrevin cannot reach its final destination and accumulates in the ER. Precisely how the function of BAP31 is affected by this deletion remains unclear. The deletion mutant still appears to be able to form a complex with cellubrevin, exhibiting properties that are not obviously different from the wild-type complex. We observed, however, that upon extended culturing cells developed abnormal membrane blebs, suggesting a delayed noxious effect of the mutant protein (Fig. , j–l
). These blebs may represent membrane accumulations at the exit site of the ER (Bannykh et al., 1996
), as suggested by the colocalization of myc
-BAPTMR with p58 in these blebs (Fig. , m–o
) or, alternatively, accumulation of membrane destined for degradation. It is possible that vesicle traffic out of the ER is affected to some extent, which may contribute to the phenotype.
We conclude that BAP31 is a representative of a novel class of proteins that regulates trafficking of certain membrane proteins out of the ER, either by retaining newly synthetized membrane proteins in the ER or by functioning as a conveyor belt for actively transporting these proteins from the ER to the Golgi complex. This class may include additional proteins such as BAP29 (Adachi et al., 1996
) and other members of the BAP family which are specific for different membrane immunoglobulins (Kim et al., 1994
; Terashima et al., 1994
). Thus, trafficking of membrane proteins by means of such control proteins may be a common mechanism in eukaryotic cells.