Interaction of the Mammalian Endosomal Sorting Complex Required for Transport (ESCRT) III Protein hSnf7-1 with Itself, Membranes, and the AAA+ ATPase SKD1

Yuan Lin,Teresa V Naismith,Lisa A Kimpler,Joshua M Lauer,Phyllis I Hanson

doi:10.1074/jbc.m413968200

Yuan Lin, Teresa V Naismith + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m413968200

Copy DOI

Abstract

SKD1/VPS4B is an AAA+ (ATPase associated with a variety of cellular activities) protein involved in multivesicular body (MVB) biogenesis. In this study, we show that the impairment in MVB biogenesis caused by the ATP hydrolysis-deficient mutant SKD1(E235Q) is accompanied by assembly of a large detergent-insoluble protein complex that includes normally soluble endogenous components of mammalian endosomal sorting complex required for transport (ESCRT) I and ESCRT-III complexes. Membrane-bound ESCRT-III complex has been proposed to be the substrate that recruits SKD1 to nascent MVBs. To explore this relationship, we studied interactions among the human ESCRT-III components hSnf7-1 and hVps24, membranes, and SKD1. We found that a significant portion of overexpressed hSnf7-1 associated with membranes where it formed a large protein complex that recruited SKD1 and perturbed normal MVB biogenesis. Overexpressed hVps24 also associated with membranes and perturbed endosome structure but only when fused to green fluorescent protein. Domain analysis revealed that the basic N-terminal half of hSnf7-1 localized to membranes and formed detergent-resistant polymers, some of which looked like filopodia extending into the lumen of swollen endosomes or out from the plasma membrane. The C-terminal acidic half of hSnf7-1 did not associate with membranes and was required for interaction of hSnf7-1 with SKD1. Together with earlier studies, our work suggests that a variety of ESCRT-III-containing polymers can assemble on membranes and recruit SKD1 during formation of the MVB.

Highlights

A network of proteins first identified in Saccharomyces cerevisiae as the class E subset of vacuolar protein sorting mutants [8] are required for internalizing proteins and membrane into the lumen of the endosome [4]
While endosomal sorting complex required for transport (ESCRT)-III proteins and the AAAϩ ATPase SKD1/ VPS4B have been implicated in multivesicular body (MVB) biogenesis in both yeast and mammalian systems, much remains to be learned about the relationships between these proteins and their effects on endosomal membranes
We studied the interaction of two human ESCRT-III proteins, hSnf7-1 and hVps24, with membranes and SKD1

Summary

Introduction

A network of proteins first identified in Saccharomyces cerevisiae as the class E subset of vacuolar protein sorting (vps) mutants [8] are required for internalizing proteins and membrane into the lumen of the endosome [4]. Elegant studies carried out over the last few years have established that class E proteins assemble into functional complexes These include the S. cerevisiae endosomal sorting complex required for transport (ESCRT) complexes I, II, and III [13,14,15] as well as a Vps27-Hse complex [16]. SKD1 and Mammalian ESCRT-III Proteins causes ESCRT components to accumulate in a large detergentresistant complex on the class E compartment membrane [14, 15, 25, 26]. This suggests that Vps is a critical regulator of proteins in the MVB pathway. Its function depends on its ability to bind and hydrolyze ATP [25] with class E defects appearing both in yeast that lack Vps and in yeast that express ATP hydrolysis-deficient Vps mutants [23, 27]

Methods

Results

Conclusion