Yeast homotypic vacuole fusion requires the Ccz1–Mon1 complex during the tethering/docking stage

Abstract

The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1–Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1–Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1–Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1–Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.

Numb regulates vesicular docking for homotypic fusion of early endosomes via membrane recruitment of Mon1b

“…Surprisingly, Mon1a, unlike Mon1b, does not play a role in EE docking. This is, however, consistent with previous finding that Mon1b, but not Mon1a, is a core component of the class C VPS/ HOPS complex [36], which is required for EE homotypic docking and fusion.…”

“…Here, we show that the 11-aa insertion in the PTB domain also determines the role of Numb in the control of endocytic trafficking. We show although all isoforms of Numb and Numblike can bind to Mon1a and Mon1b [36,47], only Numb 65, Numb 71 and partially Numblike are responsible for EE docking during homotypic fusion. In line with this finding, we observed that Numb 65, Numb 71 and partially Numblike localize on EEs in the perinuclear region, where endocytic sorting occurs in the mammalian cells.…”

“…The yeast Mon1 is crucial for vacuolar tethering/ docking [36], but the role of its mammalian homolog in EE fusion is unknown. By using RNA interference [38], we knocked down Mon1a and Mon1b ( Figure 6A and Supplementary information, Figure S9A and S9B).…”

“…We further demonstrate that Numb binds to Mon1b, a mammalian homolog of a yeast vacuolar tethering/docking factor Mon1. A mutation in yeast Mon1 impairs cis-SNARE complex assembly and the subsequent trans-SNARE pairing [36]. Loss of Numb blocks cytosolic Mon1b from being recruited to EEA1-positive vesicles.…”

“…In general, EE homotypic fusion occurs in distinct but consecutive processes, i.e., vesicular tethering, docking, and fusion, and involves multiple proteins including RabGTPases, NSF, a-SNAP, SNAP 25 and EEA1, as well as the SNARE complex [30][31][32][33][34][35]. Briefly, activated Rab5 drives NSF-primed endosomes to tether and dock with each other via oligomerized EEA1, syntaxin12/13 [32] and possibly the Mon1/CCZ1 complex [36] for subsequent homotypic fusion to generate a fused large endosome. Proteins in the fused large endosomes are either recycled back to the plasma membrane or transported to the trans-Golgi network or lysosome for destruction [37].…”