Homotypic Vacuole Fusion in Yeast Requires Organelle Acidification and Not the V-ATPase Membrane Domain

Abstract

SUMMARY Studies of homotypic vacuole-vacuole fusion in the yeast Saccharomyces cerevisiae have been instrumental in determining the cellular machinery required for eukaryotic membrane fusion and have implicated the vacuolar H+-ATPase (V-ATPase). The V-ATPase is a multi-subunit, rotary proton pump whose precise role in homotypic fusion is controversial. Models formulated from in vitro studies suggest that it is the proteolipid proton-translocating pore of the V-ATPase that functions in fusion, with further studies in worms, flies, zebrafish, and mice appearing to support this model. We present two in vivo assays and use a mutant V-ATPase subunit to establish that it is the H+-translocation/vacuole acidification function, rather than the physical presence of the VATPase that promotes homotypic vacuole fusion in yeast. Furthermore, we show that acidification of the yeast vacuole in the absence of the V-ATPase rescues vacuole fusion defects. Our results clarify the in vivo requirements of acidification for membrane fusion.

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase

“…Moreover, we demonstrate here that overexpression of AVP1 does not ameliorate the vha-a2 vha-a3 mutant phenotype, which also argues against an important role of the V-PPase in vacuolar acidification. Interestingly, the fact that in Arabidopsis lack of tonoplast V-ATPase activity cannot be compensated for by increased V-PPase activity stands in contrast to the fact that expression of AVP1 rescues vph1, a yeast mutant lacking the vacuolar isoform of the V-ATPase (Pérez-Castiñeira et al, 2011;Coonrod et al, 2013). This is most easily reconciled by assuming that the activities of V-ATPase and V-PPase depend on each other and our results concerning cold acclimation support such a scenario.…”

The vacuolar-type H+-ATPase at a glance – more than a proton pump

“…Several studies argue that the physical presence of the V 0 domain -and not the H + -pumping ability of the V-ATPaseis required for fusion (Baars et al, 2007;Bayer et al, 2003). However, a very recent study supports the original conclusion, namely that acidification, rather than forming a fusion pore is the main role of the V-ATPase in vesicle fusion (Coonrod et al, 2013). Regardless of the ultimate outcome of this controversy, the importance of the V-ATPase in fusion appears unquestionable.…”

Vacuolar ATPase in Phagosome-Lysosome Fusion

“…However, some of this work is disputed, as a recent study on homotypic vacuole fusion in yeast argues strongly that acidification rather than the presence of the v-ATPase is a major driving factor of membrane fusion (17).…”

“…The original observation of an acidification-independent role in membrane fusion arose from studies on yeast homotypic vacuole fusion (11,12). However, a recent study using the same model organelle proposed a model in which the major role of the v-ATPase complex would be for vacuolar acidification (17).…”