The Small GTPase Cdc42 Interacts with Niemann-Pick C1-like 1 (NPC1L1) and Controls Its Movement from Endocytic Recycling Compartment to Plasma Membrane in a Cholesterol-dependent Manner

Chang Xie,Na Li,Zheng-Jun Chen,Bo-Liang Li,Bao-Liang Song

doi:10.1074/jbc.m111.270199

Abstract

Niemann-Pick C1-like 1 (NPC1L1) is a multi-transmembrane protein that mediates the absorption of dietary and biliary cholesterol through vesicular endocytosis. The subcellular localization of NPC1L1 is regulated by cholesterol. Cholesterol depletion induces the transport of NPC1L1 to plasma membrane (PM) from endocytic recycling compartment that requires MyoVb·Rab11a·Rab11-FIP2 triple complex, and cholesterol-replenishment renders the internalization of NPC1L1 together with cholesterol. Here, we find that GTP-bound Cdc42 interacts with NPC1L1. Cholesterol depletion regulates the activation of Cdc42 and enhances NPC1L1-Cdc42 interaction. Overexpression of constitutive GTP-bound Cdc42 mutant form or knockdown of Cdc42 inhibits the transport of NPC1L1 to the PM and disturbs the cholesterol-regulated binding of NPC1L1 to Rab11a, MyoVb, and actin. Knockdown of Cdc42 downstream effectors N-WASP or Arp3 also leads to the similar results. In liver-specific Cdc42 knock-out (Cdc42 LKO) mice, NPC1L1 fails to localize to bile canaliculi, and the biliary cholesterol cannot be efficiently reabsorbed. These results indicate that Cdc42 controls the cholesterol-regulated transport and localization of NPC1L1, and plays a role in cholesterol absorption.

Highlights

Intestinal absorption is a major way for mammals to obtain the exogenous cholesterol, which is mainly mediated by a multi-transmembrane protein Niemann-Pick C1-like 1 (NPC1L1)2 [1, 2]
NPC1L1 Preferentially Interacts with GTP-bound Form of Cdc42—To identify proteins involved in NPC1L1-mediated cholesterol absorption, we performed a large-scale co-IP followed by SDS-PAGE and tandem mass spectrometry using the NPC1L1-EGFP cell line [9]
To further investigate whether the interaction of NPC1L1 and Cdc42 depends on the activity of Cdc42, we constructed the constitutively active (GTP-locked form, G12V) and inactive (GDP-locked form, T17N) Cdc42 mutants, and performed GST pull-down assays with GST-PAK1-PBD-agarose [20, 22,23,24]

Summary

Introduction

Intestinal absorption is a major way for mammals to obtain the exogenous cholesterol, which is mainly mediated by a multi-transmembrane protein Niemann-Pick C1-like 1 (NPC1L1)2 [1, 2]. The NPC1L1 protein localizes on the brush border membrane of small intestine and canalicular membrane of liver. It is critical for dietary cholesterol absorption and biliary cholesterol reabsorption [1, 3]. On PM, NPC1L1 binds exogenous cholesterol and form cholesterol-enriched membrane domains [8, 11]. Once bound to GTP, Cdc interacts with N-WASP and releases its autoinhibitory conformation, which binds and activates Arp2/3 complex to initiate branched actin polymerization [13, 14]. Co-immunoprecipitation (co-IP) assays demonstrate that Cdc together with N-WASP and Arp2/3 are required for cholesterol regulated binding of NPC1L1 to Rab11a, MyoVb, and actin. In vivo analyses in Cdc LKO mice indicate that depletion of Cdc disrupts canalicular localization of NPC1L1 and decreases the reabsorption of biliary cholesterol

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