Rab-Effector-Kinase Interplay Modulates Intralumenal Fragment Formation during Vacuole Fusion

Abstract

Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when the interaction between the Rab-GTPase Ypt7 and its effector Vps41 (a subunit of the tethering complex HOPS) is interrupted by a point mutation (Ypt7-D44N). Subsequent phosphorylation of Vps41 by the casein kinase Yck3 prevents stabilization of trans-SNARE complexes needed for lipid bilayer pore formation. Impairing ILF formation prevents clearance of misfolded proteins from vacuole membranes and promotes organelle permeability and cell death. We propose that HOPS coordinates Rab, kinase, and SNARE cycles to modulate ILF size during vacuole fusion, regulating lipid and protein turnover important for quality control and membrane integrity.

Sphingolipids regulate the tethering stage of vacuole fusion by affecting membrane fluidity

“…2D), and also no major time delay between lipid mixing and content mixing ( Fig. 2E), indicative of normal pore formation kinetics (Karim et al, 2018). Together, these results show C26 VLCFAs positively regulate homotypic vacuole fusion, evidenced by morphological changes in live cells, and a significant reduction in the measurable in vitro lipid-and content mixing of isolated vacuoles.…”

“…However, it should be noted that other studies have demonstrated an activation of the V-ATPase upon the addition of exogenous, shortchain PtdIns(3,5)P2 and a stabilization of the VO-V1 complex by this lipid, suggestive of a protein-lipid interaction (Li et al, 2014). There has recently been significant work in uncovering the mechanism and function of the formation of intralumenal fragments (ILFs) following homotypic vacuolar fusion (Karim et al, 2018;Mattie et al, 2017;McNally et al, 2017). The formation and visualization of ILFs is possible when stalk expansion and pore formation takes place very quickly following the formation of the hemifusion diaphragm, and when the rate of hemifusion is slowed down, the ILFs do not form.…”

“…The significant loss of small GTPases Ypt7p and Rho1p from vacuolar membranes (Fig. 3A) is intriguing, as these proteins are known to function in the tethering and docking steps of homotypic fusion Eitzen et al, 2001;Eitzen et al, 2002;Haas et al, 1995;Isgandarova et al, 2007;Jones et al, 2010;Karim et al, 2018;Lachmann et al, 2012;Langemeyer et al, 2018;Logan et al, 2010;Logan et al, 2011;Ohya et al, 1993;Price et al, 2000;Vollmer et al, 1999;Wichmann et al, 1992). N-terminal fusion proteins of GFP-Ypt7p under control of the native promoter , and constitutively expressed DsRed-Rho1p on a centromeric plasmid (this study) were expressed in live yeast.…”

“…To detect fusion proteins from whole cell extracts, TCA precipitation was performed as described in (Karim et al, 2018) Fischer)] for one hour to visualize. Primary antibodies utilized for this study were used as previously described (Bodman et al, 2015;Haas et al, 1995;Haas and Wickner, 1996;Mayer and Wickner, 1997;Peng et al, 2006;Price et al, 2000;Seals et al, 2000) Proton pumping…”

“…The Rab subfamily of small GTPases has long been known as regulators of membrane trafficking, and Ypt7p function has been well established at the vacuole (Wichmann et al, 1992) (Haas et al, 1995) (Vollmer et al, 1999) Karim et al, 2018;Lachmann et al, 2012;Langemeyer et al, 2018;Price et al, 2000). Additionally, the Rho subfamily's contributions to the fusion cascade at the yeast vacuole have been investigated, which showed they regulate actin polymerization at multiple sites within a cell (Eitzen et al, 2001;Eitzen et al, 2002;Isgandarova et al, 2007;Jones et al, 2010;Logan et al, 2010;Logan et al, 2011).…”