The mitochondria-associated membrane (MAM) is a specialized subdomain of the endoplasmic reticulum (ER) which acts as an intracellular signaling hub. MAM dysfunction has been related to liver disease. We report a high-throughput mass spectrometry-based proteomics characterization of MAMs from mouse liver, which portrays them as an extremely complex compartment involved in different metabolic processes, including steroid metabolism. Interestingly, we identified caveolin-1 (CAV1) as an integral component of hepatic MAMs, which determine the relative cholesterol content of these ER subdomains. Finally, a detailed comparative proteomics analysis between MAMs from wild type and CAV1-deficient mice suggests that functional CAV1 contributes to the recruitment and regulation of intracellular steroid and lipoprotein metabolism-related processes accrued at MAMs. The potential impact of these novel aspects of CAV1 biology on global cell homeostasis and disease is discussed.

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell‐cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol‐rich liquid‐ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post‐translational modification targets Rac1 for stabilization at actin cytoskeleton‐linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C‐terminal polybasic region and is regulated by the triproline‐rich motif. Non‐palmitoylated Rac1 shows decreased GTP loading and lower association with detergent‐resistant (liquid‐ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation‐deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1‐deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization. There is a [Have you seen?][1] (February 2012) associated with this Article. [1]: http://dx.doi.org/10.1038/emboj.2011.481

The composition and physical properties of the extracellular matrix (ECM) critically influence tumor progression, but the molecular mechanisms underlying ECM layering are poorly understood. Tumor–stroma interaction critically depends on cell communication mediated by exosomes, small vesicles generated within multivesicular bodies (MVBs). We show that caveolin-1 (Cav1) centrally regulates exosome biogenesis and exosomal protein cargo sorting through the control of cholesterol content at the endosomal compartment/MVBs. Quantitative proteomics profiling revealed that Cav1 is required for exosomal sorting of ECM protein cargo subsets, including Tenascin-C (TnC), and for fibroblast-derived exosomes to efficiently deposit ECM and promote tumor invasion. Cav1-driven exosomal ECM deposition not only promotes local stromal remodeling but also the generation of distant ECM-enriched stromal niches in vivo. Cav1 acts as a cholesterol rheostat in MVBs, determining sorting of ECM components into specific exosome pools and thus ECM deposition. This supports a model by which Cav1 is a central regulatory hub for tumor–stroma interactions through a novel exosome-dependent ECM deposition mechanism.