The formation of helical bundles of SNARE proteins from opposite membranes likely drives intracellular membrane fusion. The discovery of syntaxin 1A, a SNARE largely localized to the plasma membrane, and Sed5p (syntaxin 5 in mammalian cells) (Hardwick and Pelham 1992
; Hay and Scheller 1997
), a syntaxin isoform largely localized to the cis
-Golgi complex and ER-Golgi intermediate compartment, led to the idea that intracellular membrane trafficking and membrane fusion will be mediated by sets of SNARE proteins. Furthermore, if SNARE proteins pair specifically, part of the specificity of membrane trafficking may be mediated by the formation of specific sets of core fusion complexes. These ideas have become known as the SNARE hypothesis (Bennett et al. 1993
; Sollner et al. 1993
). For this hypothesis to be correct, several criteria must be met. First, there need to be enough SNARE proteins so that specific SNAREs, or at least a specific combination of SNAREs, could direct each vesicular trafficking step. In mammalian cells, there are in fact many different SNARE proteins, and they do have specific patterns of localization within cells. For example, syntaxin 5, rsec22b, rbet1, and membrin are important in antero- and/or retrograde trafficking between the ER and the cis
-Golgi apparatus (Hay et al. 1998
). In contrast, syntaxin 1A, VAMP-1, SNAP-25, and their closely related homologues are required for exocytosis (Hay and Scheller 1997
In this report we define a function for VAMP-7. The protein is localized to LEs, where our functional data suggest that it mediates trafficking to the lysosomes. The histochemical data are supported by both polyclonal and multiple mAbs, which strengthens the contention that the immunolocalization is specific. However, the EGF breakdown monitored in our assay is only partially inhibited by antibodies against VAMP-7. Complete inhibition of a transport step has never been observed in these types of assays. The partial inhibition may be explained in several ways. The antibodies may have access to only some of the active VAMP-7, or the antibody affinity may not be sufficient to block all SNARE interactions effectively. The antibodies used for the transport inhibition studies recognize the coil domain of VAMP-7 involved in SNARE complex formation. Although this is a domain of critical functional importance, and therefore an excellent site for function-blocking antibodies, the high stability of SNARE complexes may be such that the antibody can be displaced from VAMP-7 during complex formation. Alternatively, only a portion of the EGF may travel to a degradative compartment via a VAMP-7–mediated process. Since VAMP-7 is not present on vesicles that contain the MPR, we conclude that this SNARE is not likely to be involved in trafficking of enzymes to the lysosome.
VAMP-7 that is present on the internal vesicles of LEs is likely to be degraded. This may partially explain why only low levels of VAMP-7 are found at steady state in lysosomes. In addition, we assign a significant amount of the VAMP-7 immunoreactivity to the TGN although we have not defined the subregion of the TGN that is immunoreactive for VAMP-7. The morphological definition of the TGN may include vesicles or tubules that are more appropriately considered part of the endosomal system. Since nocodozole and BFA do not affect the localization of VAMP-7, VAMP-7–positive organelles in the TGN region are more likely to be related to endosomal compartments. Consistent with the TGN localization, some VAMP-7 may be retrieved from LEs to participate in another round of membrane transport. Our data do not rule out the possibility that VAMP-7 also has a role in a distinct TGN transport process and is not used for a single trafficking step. For example, previous studies of VAMP-7 suggest a role in transport to the apical membranes in MDCK cells (Lafont et al. 1999
). In general, the morphological and functional data we present support the idea that different trafficking steps are mediated by distinct SNARE proteins, consistent with the SNARE hypothesis.
A second criteria necessary for the SNARE hypothesis to be correct is that the SNAREs pair specifically. The full spectrum of SNAREs important in trafficking through the endosomal system to the lysosomes, including those that might pair with VAMP-7, is not yet fully defined. Recent experiments have shown that many combinations of SNAREs pair with VAMP-7 to form stable complexes in vitro (Yang et al. 1999
). Syntaxin 1A/SNAP-25, syntaxin 4/SNAP-23, and syntaxin 13/SNAP-29 all form SDS-resistant complexes with VAMP-7; the midpoint temperatures (Tm
) of the unfolding transitions for these complexes are 92°C, 88°C, and 85°C, respectively (Yang et al. 1999
). However, relatively small differences in the thermal stabilities of SNARE complexes can result in dramatic differences in the abilities of the complexes to mediate membrane fusion. Therefore, the simple formation of a complex cannot be taken as an indication of a functional complex, nor can one easily draw conclusions regarding specificity based on complex formation in vitro. Given these caveats, it is the case that syntaxin 1A and SNAP-25 form the most stable complex with VAMP-7 in studies so far. The 92°C unfolding transition for this complex is more stable than that for the unfolding transition of syntaxin 1A/SNAP-25 with their known SNARE partner, VAMP-2 (Tm
, 90°C) (Yang et al. 1999
). Therefore, it is unlikely that information for the specificity of membrane trafficking is contained within the core complex-forming helical domain of VAMP-7.
If the core complex formation is not the specificity-determining event, then why have such a large number of SNAREs, particularly within the endosomal pathways? Many SNAREs, including VAMP-7, are considerably larger than the 70 aa required for fusion complex formation. In addition, these non–core complex–forming regions are more highly divergent in aa sequence between the SNAREs expressed within a species than those sequences involved in SNARE complex formation. For example, the cytoplasmic domain of VAMP-7 is 180 aa, and the function of the NH2
-terminal 120 residues of the protein is not known. Perhaps non–core complex–forming regions of SNAREs are important for interactions with proteins that regulate aspects of vesicle targeting specificity. These interactions are likely to involve interactions with Rab proteins and their effectors, and perhaps members of the sec1p (Hata et al. 1993
; Garcia et al. 1994
; Pevsner et al. 1994
) family of syntaxin-binding proteins.
The mechanisms whereby SNAREs are localized to distinct compartments are an additional critical issue for understanding the organization of membrane compartments. The aa sequence motifs that direct SNARE localization are not well understood. It is also not clear for most SNAREs if particular coat or adaptor proteins are important in localizing the proteins to specific vesicles or matching the SNAREs to particular sets of cargo proteins. It is intriguing that VAMP-7 contains a potential adaptor protein binding motif, D/EXXXLL (aa 162–167), within the SNARE coil domain. Perhaps each of the SNAREs will contain specific sequences that direct binding to particular adaptor proteins. The small number of aa that are so far defined to be important for adaptor binding interactions makes it difficult to understand their significance simply by inspection of the aa sequences of the SNAREs. Further experiments are needed to clarify these issues.
Only when we have a complete understanding of the localizations and protein interactions of all of the membrane fusion proteins of the VAMP, syntaxin, and SNAP-25 families will we be in a position to fully consider their roles in specificity. In addition, a full definition of the complexity of SNARE protein function and organization will lead to a more precise definition of complex membrane trafficking pathways within cells.