During endocytic transport, specific integral membrane proteins are sorted into intraluminal vesicles that bud from the limiting membrane of the endosome. This process, known as multivesicular body (MVB) sorting, is important for several important biological processes. Moreover, components of the MVB sorting machinery are implicated in virus budding. During MVB sorting, a cargo protein recruits components of the MVB sorting machinery from cytoplasmic pools and these sequentially assemble on the endosome. Disassembly of these proteins and recycling into the cytoplasm is critical for MVB sorting. Vacuolar protein sorting 4 (Vps4) is an AAA (ATPase associated with a variety of cellular activities) ATPase which has been proposed to play a critical role in disassembly of the MVB sorting machinery. However, the mechanism by which it disassembles the complex is not clear. Vps4 contains an N‐terminal microtubule interacting and trafficking (MIT) domain, which has previously been shown to be required for recruitment to endosomes, and a single AAA ATPase domain, the activity of which is required for Vps4 function. In this study we have systematically characterized the interaction of Vps4 with other components of the MVB sorting machinery. We demonstrate that Vps4 interacts directly with Vps2 and Bro1. We also show that a subset of Vps4 interactions is regulated by ATP hydrolysis, and one interaction is regulated by ATP binding. Finally, we show that most proteins interact with the Vps4 MIT domain. Our studies indicate that the MIT domain has a dual role in substrate binding and recruitment to endosomes and indicate that Vps4 disassembles the MVB sorting machinery by direct effects on multiple proteins.

Hormone-sensitive lipase (HSL) is a key enzyme regulating the acute activation of lipolysis. HSL functionality is controlled by multiple phosphorylation events, which regulate its association with the surface of lipid droplets (LDs). We determined the progression and stability of HSL phosphorylation on individual serine residues both spatially and temporally in adipocytes using phospho-specific antibodies. Within seconds of ␤-adrenergic receptor activation, HSL was phosphorylated on Ser-660, the phosphorylated form appearing in the peripheral cytosol prior to rapid translocation to, and stable association with, LDs. In contrast, phosphorylation of HSL on Ser-563 was delayed, the phosphorylated protein was predominantly detected on LDs, and mutation of the Ser-659/Ser-660 site to Ala significantly reduced subsequent phosphorylation on Ser-563. Phosphorylation of HSL on Ser-565 was observed in control cells; the phosphorylated protein was translocated to LDs with similar kinetics to total HSL, and the degree of phosphorylation was inversely related to phospho-HSL Ser-563 . These results describe the remarkably rapid, sequential phosphorylation of specific serine residues in HSL at spatially distinct intracellular locales, providing new insight into the complex regulation of lipolysis.The regulation of lipid storage and lipolysis in adipocytes has important implications for the maintenance of whole body lipid homeostasis (1, 2). Dysregulation is associated with obesity and the onset of metabolic disease, insulin resistance, and type II diabetes. An important goal for the future is to design strategies that allow us to manipulate lipid storage and so control weight gain. Fundamental to a targeted approach to controlling the storage and mobilization of lipid in adipose tissue is a detailed understanding of the cellular mechanisms and machinery that regulate lipolysis. Although the key players in this process have been identified, the precise regulation of the lipolytic machinery is not yet fully understood. In this study we have begun to address this by analyzing the earliest events in the activation of lipolysis at the cellular level.Adipocytes are specialized lipid droplet (LD) 2 -laden cells that store large amounts of neutral lipid, predominantly as triglycerides (TG) (3, 4). In response to extracellular stimulation by catecholamines, adipocytes hydrolyze stored TGs to generate free fatty acids and glycerol. In rodent adipocytes, the hydrolysis of neutral lipids is tightly regulated by a series of signal transduction pathways from the G-protein-coupled ␤ 3 -adrenergic receptor that culminate at the surface of the LD (2, 5). A well characterized pathway from the ␤ 3 -adrenergic receptor results in the elevation of cAMP levels, activating cAMP-dependent protein kinase/protein kinase A (PKA), which in turn phosphorylates downstream targets, including the lipid droplet scaffold/ adaptor protein perilipin (6) and the primary diacylglycerol lipase hormone-sensitive lipase (HSL) (7). The array of phosphorylation sites p...

The adipocyte is a key regulator of mammalian metabolism. To advance our understanding of this important cell, we have used quantitative proteomics to define the protein composition of the adipocyte plasma membrane (PM) in the presence and absence of insulin. Using this approach, we have identified a high confidence list of 486 PM proteins, 52 of which potentially represent novel cell surface proteins, including a member of the adiponectin receptor family and an unusually high number of hydrolases with no known function. Several novel insulin-responsive proteins including the sodium/hydrogen exchanger, NHE6 and the collagens III and VI were also identified, and we provide evidence of PM-ER association suggestive of a unique functional association between these two organelles in the adipocyte. Together these studies provide a wealth of potential therapeutic targets for the manipulation of adipocyte function and a valuable resource for metabolic research and PM biology.

Sorting of membrane proteins into intralumenal endosomal vesicles, multivesicular body (MVB) sorting, is critical for receptor down regulation, antigen presentation and enveloped virus budding. Vps4 is an AAA ATPase that functions in MVB sorting. Although AAA ATPases are oligomeric, mechanisms that govern Vps4 oligomerization and activity remain elusive. Vps4 has an N‐terminal microtubule interacting and trafficking domain required for endosome recruitment, an AAA domain containing the ATPase catalytic site and a β domain, and a C‐terminal α helix positioned close to the catalytic site in the 3D structure. Previous attempts to identify the role of the C‐terminal helix have been unsuccessful. Here, we show that the C‐terminal helix is important for Vps4 assembly and ATPase activity in vitro and function in vivo, but not endosome recruitment or interactions with Vta1 or ESCRT‐III. Unlike the β domain, which is also important for Vps4 assembly, the C‐terminal helix is not required in vivo for Vps4 homotypic interaction or dominant‐negative effects of Vps4–E233Q, carrying a mutation in the ATP hydrolysis site. Vta1 promotes assembly of hybrid complexes comprising Vps4–E233Q and Vps4 lacking an intact C‐terminal helix in vitro. Formation of catalytically active hybrid complexes demonstrates an intersubunit catalytic mechanism for Vps4. One end of the C‐terminal helix lies in close proximity to the second region of homology (SRH), which is important for assembly and intersubunit catalysis in AAA ATPases. We propose that Vps4 SRH function requires an intact C‐terminal helix. Co‐evolution of a distinct Vps4 SRH and C‐terminal helix in meiotic clade AAA ATPases supports this possibility.