The Endosomal Na+/H+ Exchanger Contributes to Multivesicular Body Formation by Regulating the Recruitment of ESCRT-0 Vps27p to the Endosomal Membrane

Abstract

Multivesicular bodies (MVBs) are late endosomal compartments containing luminal vesicles (MVB vesicles) that are formed by inward budding of the endosomal membrane. In budding yeast, MVBs are an important cellular mechanism for the transport of membrane proteins to the vacuolar lumen. This process requires a class E subset of vacuolar protein sorting (VPS) genes. VPS44 (allelic to NHX1) encodes an endosomelocalized Na ؉ /H ؉ exchanger. The function of the VPS44 exchanger in the context of vacuolar protein transport is largely unknown. Using a cell-free MVB formation assay system, we demonstrated that Nhx1p is required for the efficient formation of MVB vesicles in the late endosome. The recruitment of Vps27p, a class E Vps protein, to the endosomal membrane was dependent on Nhx1p activity and was enhanced by an acidic pH at the endosomal surface. Taken together, we propose that Nhx1p contributes to MVB formation by the recruitment of Vps27p to the endosomal membrane, possibly through Nhx1p antiporter activity.Multivesicular bodies (MVBs) 3 are a subset of late endosomes that contain internal vesicles, permitting precursor and plasma membrane proteins to reach the lysosome or vacuole lumen for degradation or maturation, respectively. In eukaryotic cells, the MVB pathway controls various important processes such as receptor down-regulation, antigen presentation, cytokinesis, retroviral budding, and autophagy (1-6). In the budding yeast Saccharomyces cerevisiae, previous studies identified a number of genes involved in vacuolar protein sorting (VPS) (7,8). These Vps proteins function at distinct steps of protein transport between the Golgi complex and the vacuole (7,8). A subset of Vps proteins, class E proteins, is involved in MVB formation. Most class E proteins are components of five distinct complexes; Vps27p-Hse1p (also called endosomal sorting complexes required for transport-0 (ESCRT-0), -I, -II, and -III and Vps4p. These complexes are required for the formation of internal vesicles and sorting of cargo proteins to the vesicles from the endosomal membrane (3, 9). Vps27p (ESCRT-0) recruits ESCRT-I, which in turn recruits and/or activates ESCRT-II and ESCRT-III at the endosome (3, 10). Recent in vitro studies have provided new insights into the precise role of each ESCRT complex in MVB formation (11-13). ESCRT-0 initiates the MVB sorting process by recruiting ESCRT-I to the endosomal membrane and concentrating ubiquitylated MVB cargos to the vesicle budding site (12). ESCRT-I and ESCRT-II are directly involved in membrane budding by stabilizing the bud necks, whereas ESCRT-III is responsible for the cleavage of bud necks (11-13). Finally, the AAA-type ATPase Vps4p dissociates the ESCRT-III complex from the endosome (14). The sequential activity of these complexes on the cytoplasmic surface of endosomes directs the budding and fission of vesicles into the lumen of the endosome and the sequestration of cargo proteins in the vesicle. Defects in ESCRT proteins prevent internal vesicle formation and result in exagg...

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

“…1C). Six of these interactions involved either HRS, which bound four of the eight studied HCV proteins with the highest apparent affinity, or its regulator, SLC9A6 (29) (Fig. 1D).…”

“…HRS was identified as a partner of several HCV proteins, and its interactions with NS2 and NS5A with the highest apparent affinity were validated in HCV-transfected cells. NS2 bound not only HRS but also the HRS regulator, SLC9A6 (29), and the latter was also bound by core. HRS has thus emerged as a critical entry point of HCV into the ESCRT network.…”

High-Resolution pH Imaging of Living Bacterial Cells To Detect Local pH Differences

“…Intracellular pH homeostasis is fundamentally essential for living cells to maintain various cellular functions. It has been reported that intracellular compartments generate a local H + gradient within the cytoplasm (4) even though the diffusion coefficient of H + is extremely high, estimated to be on the order of 10 −7 to 10 −6  cm 2 /s (5, 6). Therefore, precise measurements of local pH around biological nanomachines are critical for understanding the role of H + in their biological activities.…”

V-ATPase, ScNhx1p and Yeast Vacuole Fusion

“…In addition to its role in vacuole fusion in yeast, ScNhx1p might also function in regulating multivesicular body (MVB) formation by the recruitment of Vps27p to the endosomal membrane through its ion transport activity (Mitsui et al, 2011). Using an MVB formation assay system, the authors have found that ScNhx1p is essential for the MVB formation in the late endosome.…”

“…Using an MVB formation assay system, the authors have found that ScNhx1p is essential for the MVB formation in the late endosome. The trafficking of the class E Vps protein Vps27p to the endosome is dependent on Nhx1p transport activity (Mitsui et al, 2011). Moreover, a recent study of a genomic screening of gene deletion mutants phenocopying nhx1D has generated a gene set involved in endosome fusion reactions (including Rab signaling and actin cytoskeleton reorganization), suggesting a post-ESCRT role of ScNhxHX1p in endosomal membrane fusion (Kallay et al, 2011).…”