ALG-2 activates the MVB sorting function of ALIX through relieving its intramolecular interaction

Sheng Sun,Jian Kuang,Joe Corvera,Gary E Gallick,Xi Zhou,Sue-Hwa Lin

doi:10.1038/celldisc.2015.18

Abstract

The modular adaptor protein ALIX is critically involved in endosomal sorting complexes required for transport (ESCRT)-mediated multivesicular body (MVB) sorting of activated epidermal growth factor receptor (EGFR); however, ALIX contains a default intramolecular interaction that renders ALIX unable to perform this ESCRT function. The ALIX partner protein ALG-2 is a calcium-binding protein that belongs to the calmodulin superfamily. Prompted by a defined biological function of calmodulin, we determined the role of ALG-2 in regulating ALIX involvement in MVB sorting of activated EGFR. Our results show that calcium-dependent ALG-2 interaction with ALIX completely relieves the intramolecular interaction of ALIX and promotes CHMP4-dependent ALIX association with the membrane. EGFR activation induces increased ALG-2 interaction with ALIX, and this increased interaction is responsible for increased ALIX association with the membrane. Functionally, inhibition of ALIX activation by ALG-2 inhibits MVB sorting of activated EGFR as effectively as inhibition of ALIX interaction with CHMP4 does; however, inhibition of ALIX activation by ALG-2 does not affect cytokinetic abscission or equine infectious anemia virus (EIAV) budding. These findings indicate that calcium-dependent ALG-2 interaction with ALIX is specifically responsible for generating functional ALIX that supports MVB sorting of ubiquitinated membrane receptors.

Highlights

EGF binding to epidermal growth factor receptor (EGFR) on the cell surface induces both the activation and endocytosis of EGFR
To determine the effect of Apoptosis-linked gene-2 product (ALG-2) interaction with ALIX on the intramolecular interaction of ALIX, we first verified the previous findings that glutathione S-transferase (GST)-ALG-2 interacts with cytosolic ALIX in the presence of 10 μM calcium (Supplementary Figure S1B), and that the E47A/E114A mutant form of ALG-2 (Mut ALG-2), which is defective in calcium binding, does not interact with cytosolic ALIX in the presence of calcium (Supplementary Figure S1C) [26]
The GST-ALG-2, but not GST, addition induced a low level of ALIX immunoprecipitation by the 2H12 antibody, whereas neither GST nor GST-ALG-2 addition changed ALIX immunoprecipitation by the 1A12 antibody. These results suggest that ALG-2 interaction with ALIX relieves the intramolecular interaction of ALIX

Summary

Introduction

EGF binding to epidermal growth factor receptor (EGFR) on the cell surface induces both the activation and endocytosis of EGFR. Endocytosis of activated EGFR does not stop the signaling function of the receptor; it poises the receptor for being sorted into the intraluminal vesicles of multivesicular body (MVB) (MVB sorting), which terminates the signaling function of activated EGFR before its eventual degradation [1]. MVB sorting of endocytosed EGFR is carried out by multiple ESCRT complexes and associated proteins. Activated EGFR is first ubiquitinated by Cbl [2, 3]. EGFR to be recognized by ESCRT-0, ESCRT-I, and ESCRT-II. The recognized EGFR is sorted into the intraluminal vesicles of MVBs through membrane invagination and scission, which is driven by ESCRT-III assembly and disassembly [4]. How activated EGFR positively regulates these processes is not fully understood

Methods

Results

Conclusion