Association of stomatin with lipid bodies

The oligomeric lipid raft-associated integral protein stomatin normally localizes to the plasma membrane and the late endosomal compartment. Similar to the caveolins, it is targeted to lipid bodies (LBs) on overexpression. Endogenous stomatin also associates with LBs to a small extent. Green fluorescent protein-tagged stomatin (StomGFP) and the dominant-negative caveolin-3 mutant DGV(cav3)(HA) occupy distinct domains on LB surfaces but eventually intermix. Studies of StomGFP deletion mutants reveal that the region for membrane association but not oligomerization and raft association is essential for LB targeting. Blocking protein synthesis leads to the redistribution of StomGFP from LBs to LysoTracker-positive vesicles indicating a connection with the late endosomal/ lysosomal pathway. Live microscopy of StomGFP reveals multiple interactions between LBs and microtubule-associated vesicles possibly representing signaling events and/or the exchange of cargo. Proteomic analysis of isolated LBs identifies adipophilin and TIP47, various lipid-specific enzymes, cytoskeletal components, chaperones, Ras-related proteins, protein kinase D2, and other regulatory proteins. The association of the Rab proteins 1, 6, 7, 10, and 18 with LBs indicates various connections to other compartments. Our data suggest that LBs are not only involved in the storage of lipids but also participate actively in the cellular signaling network and the homeostasis of lipids.

Caveolins in the repair phase of acute renal failure after oxidative stress

Ischaemia-reperfusion and toxic injury are leading causes of acute renal failure (ARF). Both of these injury initiators use secondary mediators of damage in oxygen-derived free radicals. Several recent publications about ischaemia-reperfusion and toxin-induced ARF have indicated that plasma membrane structures called caveolae, and their proteins, the caveolins, are potential participants in protecting or repairing renal tissues. Caveolae and caveolins have previously been ascribed many functions, a number of which may mediate cell death or survival of injured renal cells. This review proposes possible pathophysiological mechanisms by which altered caveolin-1 expression and localization may affect renal cell survival following oxidative stress.

GLUT4 overexpression or deficiency in adipocytes of transgenic mice alters the composition of GLUT4 vesicles and the subcellular localization of GLUT4 and insulin-responsive aminopeptidase

The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2GLUT4- Tg) mice were studied. GLUT4 amount was reduced by 80 - 95% in aP2-GLUT4-/- adipocytes and increased similar to10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase ( IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2GLUT4- Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.

Predicting function from structure: 3D structure studies of the mammalian golgi complex

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans-cisternae (2). The docking of vesicular-tubular clusters at the cis-face and the fragmentation of trans-cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans-ER functions in the delivery of ceramide to sphingomyelin synthase in the trans-Golgi membrane, for the regulated sorting via sphingolipid-cholesterol-rich domains. These structure-based predictions can now be tested using a variety of powerful cell and molecular tools.

Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.

Molecular characterization of Osh6p, an oxysterol binding protein homolog in the yeast Saccharomyces cerevisiae

Oxysterol binding protein (OSBP) and its homologs have been shown to regulate lipid metabolism and vesicular transport. However, the exact molecular function of individual OSBP homologs remains uncharacterized. Here we demonstrate that the yeast OSBP homolog, Osh6p, bound phosphatidic acid and phosphoinositides via its N-terminal half containing the conserved OSBP-related domain (ORD). Using a green fluorescent protein fusion chimera, Osh6p was found to localize to the cytosol and patch-like or punctate structures in the vicinity of the plasma membrane. Further examination by domain mapping demonstrated that the N-terminal half was associated with FM4-64 positive membrane compartments; however, the C-terminal half containing a putative coiled-coil was localized to the nucleoplasm. Functional analysis showed that the deletion of OSH6 led to a significant increase in total cellular ergosterols, whereas OSH6 overexpression caused both a significant decrease in ergosterol levels and resistance to nystatin. Oleate incorporation into sterol esters was affected in OSH6 overexpressing cells. However, Lucifer yellow internalization, and FM4-64 uptake and transport were unaffected in both OSH6 deletion and overexpressing cells. Furthermore, osh6 Delta exhibited no defect in carboxypeptidase Y transport and maturation. Lastly, we demonstrated that both the conserved ORD and the putative coiled-coil motif were indispensable for the in vivo function of Osh6p. These data suggest that Osh6p plays a role primarily in regulating cellular sterol metabolism, possibly stero transport.

Individual Palmitoyl Residues Serve Distinct Roles in H-ras Trafficking, Microlocalization, and Signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopallmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cbolesterol-dependent and cholesterol-independent microdomains. In contrast, monopallmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-pahnitate is weaker. Thus, membrane affinity of a pallmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopahnitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.

Osteopontin and related SIBLING glycoprotein genes are expressed by sertoli cells during mouse testis development

Matrix proteins play important roles in tissue morphogenesis. We have studied the expression of genes encoding the related SIBLING glycoproteins osteopontin (OPN), bone sialoprotein (BSP), and dentin matrix protein (DMP) during the development of male and female gonads during mouse embryogenesis. Opn mRNA was expressed specifically by Sertoli cells of the developing testis cords, in the mesonephric tubules of both sexes, and, transiently, in the Mullerian ducts of both sexes, as determined by whole-mount and section in situ hybridization. OPN protein was detected in the cytoplasm of Sertoli cells and luminal cells of the mesonephric tubules, with small amounts associated with the plasma membrane of germ cells. We found no defects in developing testes of Opn-/- mice using a range of cell type-specific markers, suggesting that other SIBLING proteins may function in testis development. Dmp and Bsp mRNA was also expressed in the developing testis cords, supporting the view that all three SIBLING proteins may contribute to testis differentiation. (c) 2005 Wiley-Liss, Inc.

Subcellular localization determines MAP kinase signal output

The Raf-MEK-ERK MAP kinase cascade transmits signals from activated receptors into the cell to regulate proliferation and differentiation. The cascade is controlled by the Ras GTPase, which recruits Raf from the cytosol to the plasma membrane for activation. In turn, MEK, ERK, and scaffold proteins translocate to the plasma membrane for activation. Here, we examine the input-output properties of the Raf-MEK-ERK MAP kinase module in mammalian cells activated in different cellular contexts. We show that the MAP kinase module operates as a molecular switch in vivo but that the input sensitivity of the module is determined by subcellular location. Signal output from the module is sensitive to low-level input only when it is activated at the plasma membrane. This is because the threshold for activation is low at the plasma membrane, whereas the threshold for activation is high in the cytosol. Thus, the circuit configuration of the module at the plasma membrane generates maximal outputs from low-level analog inputs, allowing cells to process and respond appropriately to physiological stimuli. These results reveal the engineering logic behind the recruitment of elements of the module from the cytosol to the membrane for activation.

Caveolin, cholesterol, and lipid bodies

In mammalian cells a complex interplay regulates the distribution of cholesterol between intracellular membrane compartments. One important aspect of cholesterol regulation is intracellular cholesterol storage in neutral lipid storage organelles called lipid droplets or lipid bodies (LBs). Recent work has thrust the LB into the limelight as a complex and dynamic cellular organelle. LBs play a crucial role in maintaining the cellular levels of cholesterol by regulating the interplay between lipid storage, hydrolysis and traffickin,-. Studies of caveolins, caveolar membrane proteins linked to lipid regulation, are providing new insights into the role of LBs in regulating cholesterol balance. (c) 2005 Elsevier Ltd. All rights reserved.

Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles

Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1 null (Cav1 -/-) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1 -/- MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveoloe and to identify noncaveolar endocytic vehicles. In WT MEFs a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2 % per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.

A role for the phagosome in cytokine secretion

Membrane traffic in activated macrophages is required for two critical events in innate immunity: proinflammatory cytokine secretion and phagocytosis of pathogens. We found a joint trafficking pathway linking both actions, which may economize membrane transport and augment the immune response. Tumor necrosis factor alpha (TNF alpha) is trafficked from the Golgi to the recycling endosome (RE), where vesicle-associated membrane protein 3 mediates its delivery to the cell surface at the site of phagocytic cup formation. Fusion of the RE at the cup simultaneously allows rapid release of TNF alpha and expands the membrane for phagocytosis.

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3 '-kinase-dependent machinery

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.