Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

Eric Bartee,Craig A Eyster,Julie G Donaldson,Klaus Früh,Mandana Mansouri,Kasinath Viswanathan,Robert E Means

doi:10.1371/journal.pone.0015132

Abstract

Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

Highlights

Ubiquitination plays a key role in regulating many diverse cellular functions, predominantly by tagging proteins for destruction via either the proteasome or lysosome
Following metabolic labeling, ‘light’ labeled samples were infected with Adenovirus expressing the Tetracycline regulatable-transactivator (Ad-Tet) alone, while ‘heavy’ labeled’ samples were infected with Ad-Tet together with Tet-induced Ad-Membrane associated RING-CH (MARCH)-VIII. 24 h post infection, cells were harvested via mechanical scraping and cellular fractions were separated as described [14] except that only the plasma membrane (PM) fraction was analyzed
Follow up experiments demonstrated that ALCAM was down-regulated by MARCH-VIII and Myxomavirus M153R whereas BST2/Tetherin was down-regulated by MARCHVIII and HIV-1 Vpu

Summary

Introduction

Ubiquitination plays a key role in regulating many diverse cellular functions, predominantly by tagging proteins for destruction via either the proteasome or lysosome. Membrane associated RING-CH (MARCH) proteins, belong to a family of transmembrane ubiquitin ligases (for a recent review see: [2]) that was initially discovered when RING-CH proteins encoded by gamma-2 herpesviruses (KSHV-K3, KSHV-K5, MHV68-K3) were shown to down-regulate the surface expression of transmembrane immune-stimulatory host cell proteins, MHC class I, contributing to viral immune evasion [3,4,5]. Leporipoxviruses encode similar MHC-I down-regulating proteins which contribute to viral virulence [6,7]. This immunoreceptor down-regulation is accomplished by the viral proteins ubiquitinating lysines in the cytoplasmic tails of their transmembrane substrates [8]. The sequence and structural homology of the viral MARCH proteins to host MARCH family suggested that the viral proteins were pirated from ancestral host proteins that likely perform related functions

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