RESUMEN
Gp78 is an E3 ubiquitin ligase within the endoplasmic reticulum-associated degradation pathway. We show that Flag-tagged gp78 undergoes sulfhydryl cysteine palmitoylation (S-palmitoylation) within the RING finger motif, responsible for its ubiquitin ligase activity. Screening of 19 palmitoyl acyl transferases (PATs) identified five that increased gp78 RING finger palmitoylation. Endoplasmic reticulum (ER)-localized Myc-DHHC6 overexpression promoted the peripheral ER distribution of Flag-gp78 while RING finger mutation and the palmitoylation inhibitor 2-bromopalmitate restricted gp78 to the central ER. Palmitoylation of RING finger cysteines therefore regulates gp78 distribution to the peripheral ER.
Asunto(s)
Retículo Endoplásmico/metabolismo , Receptores del Factor Autocrino de Motilidad/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Regulación de la Expresión Génica , Ratones , Microscopía Fluorescente/métodos , Mutación , Palmitatos/química , Ácidos Palmíticos/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi ß-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-ß-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.
Asunto(s)
Caveolas/química , Caveolina 1/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Proteoma/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Caveolas/metabolismo , Caveolina 1/genética , Línea Celular Tumoral , Endocitosis , Aparato de Golgi , Marcaje Isotópico , Microdominios de Membrana/química , Proteínas de la Membrana/genética , Ratones , N-Acetilglucosaminiltransferasas/genética , Matriz Nuclear , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genéticaRESUMEN
Phosphoglucose isomerase (PGI) is a glycolytic enzyme that exhibits a dual function as an extracellular cytokine, under the name autocrine motility factor (AMF). Its cell surface receptor, gp78/AMFR, is also localized to the endoplasmic reticulum where it functions as an E3 ubiquitin ligase. Expression of both AMF/PGI and gp78/AMFR is associated with cancer and, in this review, we will discuss various aspects of the biology of this ligand-receptor complex and its role in tumor progression.