Termination of Isoform‐Selective Vps21/Rab5 Signaling at Endolysosomal Organelles by Msb3/Gyp3

Abstract

Traffic through endosomes and lysosomes is controlled by small G-proteins of the Rab5 and Rab7 families. Like humans, Saccharomyces cerevisiae has three Rab5s (Vps21, Ypt52, and Ypt53), and one Rab7 (Ypt7). Here, we elucidate the functional roles and regulation of the yeast Rab5s. Using GFP-tagged cargoes, a novel quantitative multivesicular body (MVB) sorting assay, and electron microscopy, we show that MVB biogenesis and cargo sorting are severely impaired in vps21Δ ypt52Δ double mutants. Ypt53, the third Rab5 paralog, is hardly expressed during normal growth but its transcription is strongly induced by cellular stress through the calcineurin-Crz1 pathway. The requirement for Rab5 activity in stress tolerance facilitated identification of Msb3/Gyp3 as the principal Rab5 GAP (GTPase accelerating protein), and in vitro GAP assays verified that Vps21 is a preferred Gyp3 target. Finally, we demonstrate that Gyp3 spatially restricts active Vps21 to intermediate endosomal compartments by preventing Vps21 accumulation on lysosomal vacuoles. Gyp3 therefore operates as a compartmental insulator that enforces the boundary between penultimate and terminal compartments of the endolysosomal pathway.

Aneuploidy shortens replicative lifespan in Saccharomyces cerevisiae

“…Separate from its activity as an Rsp5 client, trafficking of Cps1 from the cell membrane to the vacuole has been well characterized and Cps1 is frequently used as a model cargo to study protein trafficking through MVBs (Odorizzi et al ., 1998). Using a quantitative, luciferase‐based assay to search for defects in MVB formation and sorting (Nickerson et al ., 2012; Nickerson & Merz, 2015), we observed that the original chr5 disome has a Cps1 trafficking defect that is ameliorated by the deletion of BUL1 or the bul1 Q146K allele (Fig. 4).…”

“…See Nickerson et al . (2012) for details (unpaired, two‐tailed t ‐test, ** P  =   0.002, *** P  =   0.0002). Bars represent means ± SD of 4 biological replicates each.…”

“…Endosomal luminal cargo transport was measured using a chimeric luciferase reporter and a dual luciferase assay (Promega) essentially as described (Nickerson et al ., 2012). Cells were grown to late log phase in selective media to ensure the retention of reporter plasmid pDN278 (Nickerson & Merz, 2015), which encodes a chimeric luminal cargo, FLuc‐CPS1 , under the native CPS1 promoter, and a soluble loading control, Renilla Luc, under the constitutive PGK1 promoter in a pDN616 backbone (Nickerson et al ., 2012).…”

“…Cells were grown to late log phase in selective media to ensure the retention of reporter plasmid pDN278 (Nickerson & Merz, 2015), which encodes a chimeric luminal cargo, FLuc‐CPS1 , under the native CPS1 promoter, and a soluble loading control, Renilla Luc, under the constitutive PGK1 promoter in a pDN616 backbone (Nickerson et al ., 2012). Measured sequentially in the same yeast cell lysate, signal from FLuc‐tagged cargo is normalized vs. signal from RLuc.…”

Bifurcation of the endocytic pathway into Rab5-dependent and -independent transport to the vacuole

“…The small GTPase, Rab5, is a key regulator of early endocytosis, being involved in targeting plasma membrane-derived endocytic vesicles to endosomes, fusion between early endosomes, MVB biogenesis and endosomal motility [14][15][16][17][18] . In addition to these traditional roles, it has recently been demonstrated that Rab5 has a role in maturation of the early to the late endosome [19][20][21] .…”