Class E compartments form in response to ESCRT dysfunction in yeast due to hyperactivity of the Vps21 Rab GTPase

Abstract

SummaryThe endosomal sorting complexes required for transport (ESCRTs) mediate the budding of intralumenal vesicles (ILVs) at late endosomes. ESCRT dysfunction causes drastic changes in endosome morphology, which are manifested in Saccharomyces cerevisiae by the formation of aberrant endosomes known as class E compartments. Except for the absence of ILVs, the mechanistic basis for class E compartment biogenesis is unknown. We used electron microscopy to examine endosomal morphology in response to transient ESCRT inactivation and recovery in yeast expressing the temperature-sensitive mutant vps4 ts allele. Our results show class E compartments accumulate fourfold the amount of membrane normally present at multivesicular bodies and that multivesicular bodies can form directly from class E compartments upon recovery of ESCRT function. We found class E compartment formation requires Vps21, which is orthologous to the Rab5A GTPase in metazoans that promotes fusion of endocytic vesicles with early endosomes and homotypic fusion of early endosomes with one another. We also determined that class E compartments accumulate GTP-bound Vps21 and its effector, the class C core vacuole/endosome tethering (CORVET). Ypt7, the yeast ortholog of Rab7 that in metazoans promotes fusion of late endosomes with lysosomes, also accumulates at class E compartments but without its effector, the homotypic fusion and protein sorting (HOPS), signifying that Ypt7 at class E compartments is dysfunctional. These results suggest that failure to complete Rab5-Rab7 conversion is a consequence of ESCRT dysfunction, which results in Vps21 hyperactivity that drives the class E compartment morphology. Indeed, genetic disruption of Rab conversion without ESCRT dysfunction autonomously drives the class E compartment morphology without blocking ILV budding.

Functional Separation of Endosomal Fusion Factors and the Class C Core Vacuole/Endosome Tethering (CORVET) Complex in Endosome Biogenesis

“…On the basis of our data, we consider it likely that CORVET drives the stacking of endosomes, although it should be kept in mind that only the Vps8 subunit localizes to the class E compartment and is sufficient to cluster multivesicular bodies (14). Interestingly, Russell et al 53 did not observe an multivesicular body sorting defect in the vps21⌬ mutant, even though we (this study) and others (50) did observe this. We consider it likely that these divergent observations are due to the weaker phenotype of the mutant.…”

“…It has been shown recently that hyperactive Rab5 homolog Vps21 can trigger the formation of stacked endosomal compartments that mimic the absence of ESCRT proteins (53). On the basis of our data, we consider it likely that CORVET drives the stacking of endosomes, although it should be kept in mind that only the Vps8 subunit localizes to the class E compartment and is sufficient to cluster multivesicular bodies (14).…”

Cellular microcompartments constitute general suborganellar functional units in cells

“…It is expected that both ESCRT and retromer should have finished their sorting events before an endosome can fuse with the lysosome. Indeed, mature endosomes are filled with vesicles and lack retromer cargo (Russell and Odorizzi , 2012 ), which was sorted into tubular carriers beforehand (Cullen , 2008 ). Interestingly, retromer also binds Rab7, which is the receptor for the HOPS fusion complex and requires this Rab for its recruitment to the endosomal membrane (Rojas et al , 2008 ;Seaman et al , 2009 ).…”

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

“…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] . Deletion of the yeast Rab5 gene, VPS21, results in accumulation of the vacuolar protein Vph1p delivered from the VPS pathway and the cell surface receptor, Ste3p, delivered from the endocytic pathway in distinct vesicles 11 , suggesting that Vps21p can function at two distinct transport steps-vacuolar transport from the TGN and the early-to-late endosome transition, before convergence of the endocytic and VPS pathways.…”