The Participation of Regulatory Lipids in Vacuole Homotypic Fusion

Abstract

In eukaryotes, organelles and vesicles modulate their contents and identities through highly regulated membrane fusion events. Membrane trafficking and fusion are carried out through a series of stages that lead to the formation of SNARE complexes between cellular compartment membranes to trigger fusion. Although the protein catalysts of membrane fusion are well characterized, their response to their surrounding microenvironment, provided by the lipid composition of the membrane, remains to be fully understood. Membranes are composed of bulk lipids (e.g., phosphatidylcholine), as well as regulatory lipids that undergo constant modifications by kinases, phosphatases, and lipases. These lipids include phosphoinositides, diacylglycerol, phosphatidic acid, and cholesterol/ergosterol. Here we describe the roles of these lipids throughout the stages of yeast vacuole homotypic fusion.

Keywords: Dgk1; Diacylglycerol; Lipin1; Pah1; Sec18; phosphatidic acid.

Figure 1, Key Figure.. The Vacuole Fusion Cycle.

Depicted are the stages of vacuole fusion. 1) Isolated vacuoles contain inactive cis-SNARE complexes composed of the 3Q-SNAREs (Vam7, Vam3, and Vti1, shades of green) and 1R-SNARE (Nyv1, red). Also pictured is a PA-bound monomeric Sec18 (mSec18, purple wedge) associated with the membrane. Pah1 activity promotes moving to the next stage while excess PA blocks the progression. 2) Hexameric Sec18 (hSec18) associates with cis-SNARE through the use of Sec17 (orange trapezoids). This is promoted by ergosterol and PI(4,5)P₂. 3) Sec18 hydrolyzes ATP to use Sec17 as a molecular wedge to separate the cis-SNARE bundle into individual proteins. Sec17 and Vam7 are released from the membrane. Individual SNAREs are shown to partially acquire secondary structure in their SNARE motifs depicted as elongated lines extending from the ovals. Sec18 is thought to re-associate with the membrane as a PA-bound protomers/monomers. 4) During vacuole docking SNAREs interact in trans and partially zipper. This is promoted by PI3P, PI4P, and PI(4,5)P₂. 5) trans-SNARE complexes continue to fully zipper leading to hemifusion. The outer leaflets of both compartments merge (purple), while the inner leaflets remain separated to prevent content mixing. This is promoted by DAG, while it is inhibited by Pl(3,5)P₂, LPC, and PA. 6) Inner leaflets fuse to fully merge the compartments into a continuous membrane and mix luminal content (purple). The post-fusion SNAREs are shown now in cis and the vacuole returns to step 1 of the cycle. (Abbreviations: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; PA, phosphatidic acid; PI3P, phosphatidylinositol 3-phosphate; PI4P, phosphatidylinositol 4-phosphate; PI(3,5)P₂, phosphatidylinositol 3,5-bisphosphate; PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; LPC, lysophosphatidylcholine;

Figure 2.. Formation of the Vertex Ring on Docked Vacuoles and Spatial Lipid-Binding Probes.

Individual dispersed vacuoles labeled with a spatial probe (red). Upon docking, vacuoles form distinct morphological features. Vacuoles become tightly apposed forming a flattened disc termed the boundary membrane. The edge of the boundary where membranes come into contact is termed the vertex ring where lipids and proteins that drive fusion become enriched (red dots). The docked vacuoles are shown in both 2D and 3D projections. Our previous work has shown that PI3P, PI(4,5)P2, DAG, and ergosterol become enriched at the vertex [17].