It has been reported that there is promiscuity and diversity in SNARE-SNARE associations.15-18
Unrestricted pairing of SNAREs by random chance in a cell where continuous membrane flux is required between dynamically evolving compartments would create tremendous disturbances in overall homeostasis, including inappropriate cargo delivery and premature protein turnover to cite a few. This makes the pathway leading to trans-SNARE complex formation a critical target for regulation.
SNARE-driven membrane fusion serves different functions at different locations in a cell, ranging from neurotransmitter release to phagosome maturation. The kinetics of each fusion event must therefore be adjusted by local factors and trans-SNARE distribution influences their fusion efficiency as we have demonstrated in our previous functional studies. Also, implies that mere existence of SNAREs on opposing membranes is not sufficient for the formation of trans-SNARE complexes. Hence, orientation of the relevant SNARE topology at the appropriate location in a cell becomes an important aspect to be governed by additional factors. The requirement for SNAREs to function as the sole determinants of fusion specificity is not absolutely critical since a number of factors, by virtue of their size, abundance or localization, can see each other upstream of trans-SNARE pairing. Faithful membrane fusion would require one or more agents to exercise stringent control over and dictate specific channelization of SNARE molecules entering into trans-complexes. Based on our previous and current studies on vacuolar fusion, the dimeric HOPS tethering complex and the activated Rab GTPase Ypt7 appear as prime candidates having an intrinsic or acquired capability to perform this job.
Various lines of evidence seem to support our general idea that control over trans-SNARE complex formation occurs through collaborative action of tethers and Rabs, and that interactions among different tethering factors and the corresponding Rab proteins sets up the stage for SNARE-driven fusion. In the context of physiological membranes the vesicle tethering protein p115 was biochemically shown to selectively catalyze the assembly of Gos28 (v-SNARE) and syntaxin5 (t-SNARE) during NSF-driven Golgi reassembly. The syntaxin binding SM(Sec1/Munc18) family member Sly1 also coprecipitated in this complex.19
Another study demonstrated a Rab1-mediated recruitment of p115 to COPII vesicles on which the cis-complex of cognate v-SNAREs was stabilized.20
In these reports, however, trans-SNARE complex formation and topology were not explicitly assayed. Studies on heterotypic fusion involving ER-derived COPII vesicles and the Golgi hint toward a QbQcR-Qa trans-SNARE distribution with the Qa SNARE acting from the Golgi membrane although each membrane contains the full complement of SNAREs.21
The asymmetry in SNARE function was suggested to be caused by an asymmetric requirement for functional Rab GTPase Ypt1 and the SM family protein Sly1 specifically on the Golgi compartment suggesting a possible role of these factors in contributing to fusion specificity.
Purified HOPS, when added to SNARE-reconstituted proteoliposomes, was recently shown to accelerate their fusion22
and HOPS was proposed to be the direct agent of tethering, but evidence for a pathway of HOPS-mediated trans-SNARE complex formation is lacking. It was shown using a liposome-based system that an endosomal Rab GTPase can dimerize in trans with itself to tether membranes.23
However, in contrast to typical multisubunit or elongated coiled-coil tethering factors, the scope for small G proteins to function as tethering agents solely by themselves appears rather limited by their overall size and surface area available for orchestrating multifarious sequential interactions. Studies involving purified SNAREs, HOPS and Rabs reconstituted into synthetic liposomes do provide critical clues about probable functions of these fusion elements along with mechanistic insights.24-26
However, to be able to firmly establish and extrapolate fundamental mechanisms underlying membrane fusion, evidence from model systems incorporating physiological membranes is essential.
Studies on rat liver Golgi membranes have shown that the coiled-coil tether Golgin-84 on COPI vesicles interacts with the cis-Golgi localized heterooctameric COG (conserved oligomeric Golgi) complex through its Cog7 subunit and it was suggested that this tether-tether interaction in trans may somehow aid SNARE complex formation.27
EEA1(early endosomal antigen 1), the rod-like coiled coil tether functioning on endosomes was shown to form a parallel coiled coil dimer. Importantly, EEA1 (both recombinant and that derived from rat brain) can be crosslinked to yield a product with double the molecular mass of the monomer. It contains an N-terminal Zinc finger, a C-terminal PI(3)P binding FYVE domain and two binding sites for a GTP-bound form of Rab5—one at the N-terminal and other at the C-terminal. It was postulated in this study that the EEA1 dimer was likely to link Rab5-enriched compartments to each other.28
TRAPPII is a tethering complex composed of ten different subunits localized at the Golgi network. Native TRAPPII purified from yeast was found to exist predominantly in dimeric form as judged by the 2-fold symmetry apparent in negatively stained electron micropraphs.29
Also gel filtration studies have shown that TRAPPII elutes at approximately twice the value of molecular masses of its individual components.30
However, it remains to be determined whether this TRAPPII dimer is able to link native membranes via interactions with Rab GTPases or SNAREs.
Experiments on HOPS subunits implicate Vps39 and Vps41 in Rab recognition and emphasize that Vps11 functions as a platform for HOPS complex assembly with its C-terminal RING domain being functionally critical for traffic to the vacuole.31
A recent advance illustrated the cryo-EM structure of the HOPS complex stating that HOPS is a flexible, seahorse-like structure, 28–35 nm long, and was not found to exist as a dimer.32
To account for previously reported interactions and modes of action of HOPS, it was suggested that HOPS could oscillate between different conformations that bring its head (Vps41, Vps33) and tail (Vps39) subunits closer or farther to accommodate binding partners, especially Ypt7 and SNAREs. We conceptualize that a HOPS dimer assimilating this behavior can indeed sequentially stabilize a QbQcR cis-SNARE complex (likely on one HOPS complex in the dimer); execute, in concert with Ypt7 from trans, long-range membrane recognition and tethering over tens of nanometers (likely through Vps39/Vps41 on the partner HOPS complex in the dimer); followed by topologically regulated QbQcR-Qa trans-SNARE complex assembly (likely through Vps33 as suggested in Bröcker et al.32
According to our homotypic model depicted in , a fusion reaction starts out with identical fusion partners each possessing a full complement of the tetrameric cis-SNARE complex. Upon Sec18/NSF-induced activation of SNAREs in the presence of ATP, a HOPS dimer, accompanied by activated Ypt7 in cis, coordinates a stable QbQcR cis-SNARE complex having displaced the Qa SNARE. The HOPS dimer recognizes activated Ypt7 in trans and incorporates the single Qa SNARE from the apposed fusion partner ultimately catalyzing Rab-dependent assembly of a QbQcR-Qa trans-SNARE complex.
Figure 4. Working model for trans-SNARE complex establishment in homotypic fusion. Each fusion partner (vacuole) is exactly identical and possesses similar fusion machinery including four vacuolar SNAREs, a HOPS dimer and Ypt7. In the presence (more ...)
Similarly a model explaining trans-SNARE complex formation in a more general heterotypic scenario can be proposed as shown in . Each fusion partner possesses its own distinct fusion machinery consisting of SNAREs, tethering factors and Rab proteins. Here, initial recognition of apposed membranes is likely to occur through interaction between their cognate tethering complexes in trans. Functional Rab proteins are required on both membranes for the heterogeneous tether dimer to be able to coordinate trans-SNARE complex assembly. Since the tethers and Rabs are already dissimilar on each fusion partner as opposed to homotypic fusion, asymmetry in SNARE pairing becomes intrinsic. Eventually the tether dimer organizes a QbQcR-Qa trans-SNARE complex leading to fusion.
Figure 5. Proposed model for trans-SNARE complex establishment in heterotypic fusion. Each fusion partner possesses its own distinct set of fusion factors. Initial recognition between the non-identical fusion partners (vesicle and plasma membrane (more ...)
Altogether our results suggest that both recognition of the appropriate fusion partner as well as coordination of a preferred trans-SNARE topology by a common Rab GTPase-dependent tether-tether scaffold may be a general mechanism that leads to homotypic and heterotypic fusion events.