Novel lipid regulation of vacuolar fusion in saccharomyces cerevisiae
Intracellular trafficking is essential for proper maturation and functioning of eukaryotic cells. The terminal stage of trafficking is the membrane fusion of two distinct compartments leading to the mixing of contents. In order to regulate this process organelles are enriched with specific fusion proteins termed SNAREs. While the core fusion machinery is well understood, relatively little is known about the role lipid regulation plays in membrane fusion. It has been well established that enrichment of specific regulatory lipids leads to recruitment and activation of the fusion machinery. Additionally these lipids are modified by specific kinases, phosphatases and lipases allowing regulation of the different stages of fusion. Furthermore, some regulatory lipids have been found to enhance the fission process. Taken together, it becomes evident that the dynamic lipid remodeling of membranes plays a key role in both membrane fusion and fission. In order to study in vitro membrane fusion with a system containing endogenous proteins and lipid, the vacuole from Saccharomyces cerevisiae is utilized. Yeast vacuoles undergo homotypic vacuolar fusion which is utilized to maintain homeostasis and occurs during inheritance. During the fusion pathway, vacuoles accumulate the glycerophospholipid phosphatidylinositol 3-phosphate (PI3P). PI3P is a positive regulatory of fusion that functions through the recruitment of multiple components of the vacuolar fusion machinery including the soluble SNARE Vam7. Vam7 interacts with PI3P through its PX domain and utilizes this interaction for proper localization to the vacuole. In Chapter 2 I show that the interaction of Vam7 with PI3P is allosterically regulated by the Vam7 Mid-domain (MD). I identified a polybasic region in the MD and found that mutating these basic residues to alanine, generating Vam7-6A, led to impaired vacuolar fusion. This was due to decreased association with its cognate SNAREs as well as the HOPS tethering complex. The reduced protein binding by Vam7-6A was not due to reduced anionic lipid binding as initially hypothesized but was associated with enhanced PI3P binding through Vam7’s PX domain. I now posit that binding PI3P with higher affinity allosterically inhibits binding to partner proteins. It can therefore be concluded that PI3P enrichment is necessary to initially trigger the fusion process through recruitment of fusion factors, but either the turnover of PI3P or the release of fusion proteins from PI3P is necessary for fusion to proceed. While enrichment of lipids can lead to organelle specific regulation, there are lipid regulation mechanisms shared throughout the cell. Of specific interest is the lipid phosphatidic acid (PA) and diacylglycerol (DAG) which are interconverted by the PA phosphatase Pah1 and the DAG kinase Dgk1. Previous work showed DAG acts as a positive regulator of vacuolar fusion and is proposed to reduce the force requirements of vacuolar fusion. I hypothesized that deletion of Dgk1 would lead to enhanced levels of DAG leading to enhanced fusion. In Chapter 3 I show that deletion of Dgk1 causes a significant enhancement in fusion and leads to a reduction in the force requirements for membrane fusion. However addition of DAG alone was unable to fully reproduce the Dgk1 deletion phenotype suggesting a reduction in PA was in part responsible for the overall fusion enhancement. In addition to PI3P, the vacuole is also highly enriched in phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a lipid shown to trigger vacuole fission. In Chapter 4 I show PI(3,5)P2 acts as a novel inhibitor of Ca2+ efflux during vacuolar fusion. I hypothesized that this inhibitory activity was through regulation of the Ca2+ efflux channel Yvc1. Surprisingly our study showed PI(3,5)P2 acts through activation of the Ca2+ influx channel Pmc1 rather than inhibition of Yvc1. This is in direct opposition to PI(3,5)P2’s role as an activator of Yvc1 in membrane fission. Taken together these findings demonstrate the complex nature of membrane fusion regulation by lipids and hopefully will serve as the basis for further investigation.
Vacuole Fusion, Phosphatidylinositol 3-phosphate, Phosphatidylinositol 3,5-bisphosphate, membrane fusion, lipid, SNARE, Membrane fluidity, Calcium transport
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign