The SNARE Motif Contributes to rbet1 Intracellular Targeting and Dynamics Independently of SNARE Interactions

Abstract

The endoplasmic reticulum/Golgi SNARE rbet1 cycles between the endoplasmic reticulum and Golgi and is essential for cargo transport in the secretory pathway. Although the quaternary SNARE complex containing rbet1 is known to function in membrane fusion, the structural role of rbet1 is unclear. Furthermore, the structural determinants for rbet1 targeting and its cyclical itinerary have not been investigated. We utilized protein interaction assays to demonstrate that the rbet1 SNARE motif plays a structural role similar to the carboxyl-terminal helix of SNAP-25 in the synaptic SNARE complex and demonstrated the importance to SNARE complex assembly of a conserved salt bridge between rbet1 and sec22b. We also examined the potential role of the rbet1 SNARE motif and SNARE interactions in rbet1 localization and dynamics. We found that, in contrast to what has been observed for syntaxin 5, the rbet1 SNARE motif was essential for proper targeting. To test whether SNARE interactions were important for the targeting function of the SNARE motif, we used charge repulsion mutations at the conserved salt bridge position that rendered rbet1 defective for binary, ternary, and quaternary SNARE interactions. We found that heteromeric SNARE interactions are not required at any step in rbet1 targeting or dynamics. Furthermore, the heteromeric state of the SNARE motif does not influence its interaction with the COPI coat or efficient recruitment onto transport vesicles. We conclude that protein targeting is a completely independent function of the rbet1 SNARE motif, which is capable of distinct classes of protein interactions.

Sec22p Export from the Endoplasmic Reticulum Is Independent of SNARE Pairing

“…2 Bet1p also binds directly to Sec24p, and its COPII binding motif is occluded when assembled into SNARE complexes, suggesting that Bet1p is also packaged into COPII vesicles in a free form (44). As for mammalian ER/Golgi SNARE proteins, heteromeric SNARE interactions are not required at any step in rbet1 targeting or transport dynamics (47). Moreover, antibodies to the mammalian ER/Golgi SNARE proteins (i.e.…”

A novel multiprotein complex is required to generate the prechylomicron transport vesicle from intestinal ER

“…6 ), antibodies to budding components VAMP7 and apoB48 were incubated with intestinal ER, the excess antibodies removed by washing, and the ER used in a budding assay. The results were compared with ER budding activity where the ER was incubated with either IgG or antibodies to ER components Bet1 and Sec22b, which are v-SNAREs for ER-to-Golgi transport vesicles other than PCTV ( 32 ). When the ER was incubated with antibodies to apoB48 or VAMP7, PCTV production was severely attenuated…”

Dynamic transport of SNARE proteins in the Golgi apparatus

“…One of the best characterized SNARE complexes is composed of Bet1, ERS24, membrin, and syntaxin 5 (1). It controls the fusion of endoplasmic reticulum (ER)-derived vesicles with the cis-Golgi, and it is generally believed that Bet1 is the v-SNARE subunit in this complex (1), although some have proposed that ERS24 might be the v-SNARE (2,3). At least one other SNARE complex (GS15-GOS28-syntaxin5-Ykt6) is present in the Golgi apparatus, but this complex is more concentrated on the trans face and might control intra-Golgi transport (either anterograde or retrograde) (4), or possibly transport between the Golgi apparatus and endosomes (5).…”

“…Efficient retrograde transport of Bet1 might be sufficient to prevent further transport along the Golgi stack, but the absence of Bet1 in trans-Golgi cisternae might also be caused by its exclusion from anterograde transport vesicles. Interestingly, it seems that the localization of Bet1 in the cis-Golgi may not be determined by its interaction with its cognate SNARE proteins (3).…”