Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRPexpressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells. Eukaryotes store lipid in cytosolic lipid droplets, which consist of neutral lipid cores surrounded by phospholipid monolayers (1-3). In mammals, lipid droplets are most abundant in adipose tissue, where stored triacylglycerol (TAG) provides the primary energy reserve for the organism. Lipid droplets in steroidogenic cells contain cholesteryl esters used in the synthesis of steroid hormones. Most other mammalian cells contain smaller lipid droplets, whose function remains unclear. They may serve as local energy reserves or sources of lipid for membrane synthesis. Furthermore, they may protect cells from the harmful effects of excess lipid accumulation by sequestering toxic lipid species away from pathways that lead to cell death (4, 5).Mechanisms controlling the synthesis and turnover of lipid droplets are only partially understood. According to one model of lipid droplet biogenesis, newly synthesized neutral lipids accumulate inside the endoplasmic reticulum membrane, forming a disk that eventually buds into the cytoplasm surrounded by an endoplasmic reticulumderived phospholipid monolayer (2, 6). Conversely, lipid droplet turnover occurs via the hydrolysis of stored neutral lipids by cytosolic lipases. Much of what we know about the regulation of lipolysis stems from studies in adipocytes. In response to hormone stimulation, protein kinase A phosphorylates two key substrates: hormone-sensitive lipase (HSL) (7) and perilipins (8, 9). Phosphorylation of HSL stimulates both its activity and its association with lipid droplets, in a manner that depends on perilipins.Perilipins regulate TAG hydrolysis in two...

Summary Mammalian mtDNA encodes only 13 proteins, all essential components of respiratory complexes, synthesized by mitochondrial ribosomes. Mitoribosomes contain greatly truncated RNAs transcribed from mtDNA, including a structural tRNA in place of 5S RNA as a scaffold for binding 82 nucleus-encoded proteins, mitoribosomal proteins (MRPs). Cryoelectron microscopy (cryo-EM) studies have determined the structure of the mitoribosome, but its mechanism of assembly is unknown. Our SILAC pulse-labeling experiments determine the rates of mitochondrial import of MRPs and their assembly into intact mitoribosomes, providing a basis for distinguishing MRPs that bind at early and late stages in mitoribosome assembly to generate a working model for mitoribosome assembly. Mitoribosome assembly is a slow process initiated at the mtDNA nucleoid driven by excess synthesis of individual MRPs. MRPs that are tightly associated in the structure frequently join the complex in a coordinated manner. Clinically significant MRP mutations reported to date affect proteins that bind early on during assembly.

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc-and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly proteinprotein interactions, target LAT to rafts.Recent years have seen an explosion of interest in membrane microdomains called lipid rafts (1-3). Rafts have been implicated in processes as diverse as signal transduction (1, 4), membrane trafficking (5, 6), and apoptosis (7). In addition, many pathogenic viruses and bacteria hijack host cell rafts during infection (8 -10). In all of these cases, function depends on selective enrichment of a subset of membrane proteins in rafts. For this reason, it is important to determine how proteins are targeted to rafts.A key feature of raft structure is the tight packing of lipid acyl chains. Raft lipids are probably in the liquid-ordered (l o ) 1 phase, in which lipid acyl chains are extended and ordered (11,12). Many proteins are targeted to rafts by their favorable association with these ordered lipids. For example, raft proteins such as glycosylphosphatidylinositol-anchored proteins, Src family kinases, and heterotrimeric G protein ␣ subunits are linked to saturated acyl chains, which partition well into rafts. Because of this tight acyl chain packing, raft lipids and proteins are insoluble in nonionic detergents and can be isolated from cell lysates as DRMs. Although DRM association of a protein may not provide a quantitative measure of its asso...

The epidermal growth factor (EGF)-receptor family member ErbB2 is commonly overexpressed in human breast cancer cells and correlates with poor prognosis. Geldanamycin (GA) induces the ubiquitylation, intracellular accumulation and degradation of ErbB2. Whether GA stimulates ErbB2 internalization is controversial. We found that ErbB2 was internalized constitutively at a rate that was not affected by GA in SK-BR-3 breast cancer cells. Instead, GA treatment altered endosomal sorting, causing the transport of ErbB2 to lysosomes for degradation. In contrast to earlier work, we found that ErbB2 internalization occurred by a clathrin-and tyrosine-kinaseindependent pathway that was not caveolar, because SK-BR-3 cells lack caveolae. Similar to cargo of the glycosylphosphatidylinositol (GPI)-anchored protein-enriched early endosomal compartment (GEEC) pathway, internalized ErbB2 colocalized with cholera toxin B subunit, GPI-anchored proteins and fluid, and was often seen in short tubules or large vesicles. However, in contrast to the GEEC pathway in other cells, internalization of ErbB2 and fluid in SK-BR-3 cells did not require Rho-family GTPase activity. Accumulation of ErbB2 in vesicles containing constitutively active Arf6-Q67L occurred only without GA treatment; Arf6-Q67L did not slow transport to lysosomes in GA-treated cells. Further characterization of this novel clathrin-, caveolae-and Rhofamily-independent endocytic pathway might reveal new strategies for the downregulation of ErbB2 in breast cancer. Supplementary material available online at

We found that PTRF/cavin-1 is lost coordinately with caveolin-1 in some cancer cells. When reexpressed in these cells, caveolin-1 formed membrane tubules that were under actomyosin-induced tension and recruited Rab8 and EHD proteins. PTRF/cavin-1 inhibited tubule formation by caveolin-1, showing a new function for the protein.