Donor Membranes Research Articles

Chondrogenic potential of human synovial mesenchymal stem cells in alginate

In a recent study, we demonstrated that mesenchymal stem cells (MSCs) derived from the synovial membranes of bovine shoulder joints could differentiate into chondrocytes when cultured in alginate. The purpose of the present study was to establish the conditions under which synovial MSCs derived from aging human donors can be induced to undergo chondrogenic differentiation using the same alginate system. MSCs were obtained by digesting the knee-joint synovial membranes of osteoarthritic human donors (aged 59-76 years), and expanded in monolayer cultures. The cells were then seeded at a numerical density of 4x10(6)/ml within discs of 2% alginate, which were cultured in serum-containing or serum-free medium (the latter being supplemented with 1% insulin, transferrin, selenium (ITS). The chondrogenic differentiation capacity of the cells was tested by exposing them to the morphogens transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2) and BMP-7, as well as to the synthetic glucocorticoid dexamethasone. The relative mRNA levels of collagen types I and II, of aggrecan and of Sox9 were determined quantitatively by the real-time polymerase chain reaction (PCR). The extracellular deposition of proteoglycans was evaluated histologically after staining with Toluidine Blue, and that of type-II collagen by immunohistochemistry. BMP-2 induced the chondrogenic differentiation of human synovial MSCs in a dose-dependent manner. The response elicited by BMP-7 was comparable. Both of these agents were more potent than TGF-beta1. A higher level of BMP-2-induced chondrogenic differentiation was achieved in the absence than in the presence of serum. In the presence of dexamethasone, the BMP-2-induced expression of mRNAs for aggrecan and type-II collagen was suppressed; the weaker TGF-beta1-induced expression of these chondrogenic markers was not obviously affected. We have demonstrated that synovial MSCs derived from the knee joints of aging human donors possess chondrogenic potential. Under serum-free culturing conditions and in the absence of dexamethasone, BMP-2 and BMP-7 were the most potent inducers of this transformation process.

The Brefeldin A-inhibited Guanine Nucleotide-exchange Protein, BIG2, Regulates the Constitutive Release of TNFR1 Exosome-like Vesicles

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.

Effect of intact donor cell membrane and cytoplasm of cumulus on development of mouse somatic clones in vitro

Reconstructed embryos can be got by two nuclear transfer methods, fusion and microinjection. However, the fusion method is time-taking and has low efficiency. Microinjection involved the donor nucleus isolation, which may lead to the damage of the nucleus DNA. Cloning efficiency may be reduced by low fusion rate of the cell fusion method and damaged DNA of microinjection method. So some researchers used the intracytoplasmic injection of the whole-cell donor, which was simple and less labor-intensive for cloning study. Here we used the intact cumulus cell as donor to study the feasibility of the microinjection method in mouse nuclear transfer. After the whole cell injection of the cumulus, most of the couplets were fragmented following activation at 1 or 6 h after injection. Whereas those only the nucleus after isolation from the cumulus was injected cleaved and developed normally. The control group of oocytes, which were not injected with the donor, was also fragmented after enucleation. Most of the couplets of the parthenotes after intact cumulus cell injection were fragmented. The group of those in which the donor was just broken and then injected (nucleus with most of the broken membrane and the cytoplasm were injected) developed as a lower rate compared to those only isolated nucleus being injected. These results suggest that the intact donor membrane of cumulus cell hinder the reprogramming of oocyte for somatic nucleus and components of donor cytoplasm and membrane affected the development of the clones in vitro.

γ2-Adaptin, a Novel Ubiquitin-interacting Adaptor, and Nedd4 Ubiquitin Ligase Control Hepatitis B Virus Maturation

Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular gamma2-adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered gamma2-adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same gamma2-adaptor and that disruption of the HBV/gamma2-adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of gamma2-adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both gamma2-adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with gamma2-adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.

TbVps34, the Trypanosome Orthologue of Vps34, Is Required for Golgi Complex Segregation

Phosphoinositides are important regulators of numerous cellular functions. The yeast class III phosphatidylinositol 3-kinase Vps34p, and its human orthologue hVPS34, are implicated in control of several key pathways, including endosome to lysosome transport, retrograde endosome to Golgi traffic, multivesicular body formation, and autophagy. We have identified the Vps34p orthologue in the African trypanosome, TbVps34. Knockdown of TbVps34 expression by RNA interference induces a severe growth defect, with a post-mitotic block to cytokinesis accompanied by a variety of morphological abnormalities. GFP2xFYVE, a chimeric protein that specifically binds phosphatidylinositol 3-phosphate, localizes to the trypanosome endosomal system and is delocalized under TbVps34 RNA interference (RNAi), confirming that TbVps34 is an authentic phosphatidylinositol 3-kinase. Expression of GFP2xFYVE enhances the TbVps34 RNAi-associated growth defect, suggesting a synthetic interaction via competition for phosphatidylinositol 3-phosphate-binding sites with endogenous FYVE domain proteins. Endocytosis of a fluid phase marker is unaffected by TbVps34 RNAi, but receptor-mediated endocytosis of transferrin and transport of concanavalin A to the lysosome are both impaired, confirming a role in membranous endocytic trafficking for TbVps34. TbVps34 knockdown inhibits export of variant surface glycoprotein, indicating a function in exocytic transport. Ultrastructural analysis revealed a highly extended Golgi apparatus following TbVps34 RNAi, whereas expression of the Golgi marker red fluorescent protein-GRASP (Grp1 (general receptor for phosphoinositides-1)-associated scaffold protein) demonstrated that trypanosomes are able to duplicate the Golgi complex but failed to complete segregation during mitosis, despite faithful replication and segregation of basal bodies and the kinetoplast. These observations implicate TbVps34 as having a role in coordinating segregation of the Golgi complex at cell division.

Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.

Macromolecular assemblies regulate nonvesicular phosphatidylserine traffic in yeast

PtdSer (phosphatidylserine) is synthesized in the endoplasmic reticulum and the related MAM (mitochondria-associated membrane), and transported to the PtdSer decarboxylases, Pds1p in the mitochondria, and Psd2p in the Golgi. Genetic and biochemical analyses of PtdSer transport are now revealing the role of specific protein and lipid assemblies on different organelles that regulate non-vesicular PtdSer transport. The transport of PtdSer from MAM to mitochondria is regulated by at least three genes: MET30 (encoding a ubiquitin ligase), MET4 (encoding a transcription factor), and one or more unknown genes whose transcription is regulated by MET4. MET30-dependent ubiquitination is required for the MAM to function as a competent donor membrane and for the mitochondria to function as a competent acceptor membrane. Non-vesicular transport of PtdSer to the locus of Psd2p is under the control of at least three genes, STT4 [encoding Stt4p (phosphatidylinositol 4-kinase)], PSTB2 (encoding the lipid-binding protein PstB2p) and PSD2 (encoding Psd2p). Stt4p is proposed to produce a pool of PtdIns4P that is necessary for lipid transport. PstB2p and Psd2p must be present on the acceptor membrane for PtdSer transport to occur. Psd2p contains a C2 (Ca(2+) and phospholipid binding sequence) domain that is required for lipid transport. Reconstitution studies with chemically defined donor membranes demonstrate that membrane domains rich in the anionic lipids, PtdSer, PtdIns4P and phosphatidic acid function as the most efficient donors of PtdSer to Psd2p. The emerging view is that macromolecular complexes dependent on protein-protein and protein-lipid interactions form between donor and acceptor membranes and serve to dock the compartments and facilitate phospholipid transport.

The cellular functions of clathrin

Membranes and proteins are moved around the cell in small vesicles. A protein coat aids the budding of such vesicles from donor membranes. The major type of coat used by the cell is composed of clathrin, a three-legged protein that can form lattice-like coats on membranes destined for trafficking. In this review, I outline what we know about clathrin and discuss some recent advances in understanding the basic biology of this fascinating molecule, which include building a molecular model of a clathrin lattice and discovery of a new function for clathrin that occurs during mitosis.

SH3P7/mAbp1 deficiency leads to tissue and behavioral abnormalities and impaired vesicle transport

The intracellular adaptor protein SH3P7 is the mammalian ortholog of yeast actin-binding protein 1 and thus alternatively named as mAbp1 (or HIP55). Structural properties, biochemical analysis of its interaction partners and siRNA studies implicated mAbp1 as an accessory protein in clathrin-mediated endocytosis (CME). Here, we describe the generation and characterization of mice deficient for SH3P7/mAbp1 owing to targeted gene disruption in embryonic stem cells. Mutant animals are viable and fertile without obvious deficits during the first weeks of life. Abnormal structure and function of organs including the spleen, heart, and lung is observed at about 3 months of age in both heterozygous and homozygous mouse mutants. A moderate reduction of both receptor-mediated and synaptic endocytosis is observed in embryonic fibroblasts and in synapses of hippocampal neurons, respectively. Recycling of synaptic vesicles in hippocampal boutons is severely impaired and delayed four-fold. The presynaptic defect of SH3P7/mAbp1 mouse mutants is associated with their constricted physical capabilities and disturbed neuromotoric behaviour. Our data reveal a nonredundant role of SH3P7/mAbp1 in CME and places its function downstream of vesicle fission.

Tetraploid and Hexaploid Wheats Express Identical Isoforms of nsLTP1

Nonspecific lipid-transfer proteins (nsLTPs) have been recognized as allergens in several plant species among which are cereals important in human nutrition. In this report, we purified a 9600 +/- 1 Da protein from both soft wheat and farro bran. Mass spectrometric analyses revealed that these proteins are identical, belong to the nsLTP1 class, and have high sequence homology with nsLTP1 isolated from other cereal species. Their identification was further supported by the ability of the soft wheat nsLTP1 to transfer pyrene-labeled lipids between donor and acceptor membranes. The results are discussed in view of the increasing diffusion on the markets of bran-rich products.

Domains within the GARP Subunit Vps54 Confer Separate Functions in Complex Assembly and Early Endosome Recognition

Tethering complexes contribute to the specificity of membrane fusion by recognizing organelle features on both donor and acceptor membranes. The Golgi-associated retrograde protein (GARP) complex is required for retrograde traffic from both early and late endosomes to the trans-Golgi network (TGN), presenting a paradox as to how a single complex can interact specifically with vesicles from multiple upstream compartments. We have found that a subunit of the GARP complex, Vps54, can be separated into N- and C-terminal regions that have different functions. Whereas the N-terminus of Vps54 is important for GARP complex assembly and stability, a conserved C-terminal domain mediates localization to an early endocytic compartment. Mutation of this C-terminal domain has no effect on retrograde transport from late endosomes. However, a specific defect in retrieval of Snc1 from early endosomes is observed when recycling from late endosomes to the Golgi is blocked. These data suggest that separate domains recruit tethering complexes to different upstream compartments to regulate individual trafficking pathways.

Structure of doubly prenylated Ypt1:GDI complex and the mechanism of GDI-mediated Rab recycling

In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.

Biogenesis of Tubular ER-to-Golgi Transport Intermediates

Tubular transport intermediates (TTIs) have been described as one class of transport carriers in endoplasmic reticulum (ER)-to-Golgi transport. In contrast to vesicle budding and fusion, little is known about the molecular regulation of TTI synthesis, transport and fusion with target membranes. Here we have used in vivo imaging of various kinds of GFP-tagged proteins to start to address these questions. We demonstrate that under steady-state conditions TTIs represent approximately 20% of all moving transport carriers. They increase in number and length when more transport cargo becomes available at the donor membrane, which we induced by either temperature-related transport blocks or increased expression of the respective GFP-tagged transport markers. The formation and motility of TTIs is strongly dependent on the presence of intact microtubules. Microinjection of GTPgammaS increases the frequency of TTI synthesis and the length of these carriers. When Rab proteins are removed from membranes by microinjection of recombinant Rab-GDI, the synthesis of TTIs is completely blocked. Microinjection of the cytoplasmic tails of the p23 and p24 membrane proteins also abolishes formation of p24-containing TTIs. Our data suggest that TTIs are ER-to-Golgi transport intermediates that form preferentially when transport-competent cargo exists in excess at the donor membrane. We propose a model where the interaction of the cytoplasmic tails of membrane proteins with microtubules are key determinants for TTI synthesis and may also serve as a so far unappreciated model for aspects of transport carrier formation.

Protein and lipid motifs regulate phosphatidylserine traffic in yeast

Phosphatidylserine (PtdSer) is synthesized in the endoplasmic reticulum and its subdomains associated with the mitochondria [MAM (mitochondria-associated membrane)] and subsequently transported to the loci of the PtdSer decarboxylases, Pds1p (phosphatidylserine decarboxylase 1 encoded by the PSD1 gene that complements psd1 mutations) in the mitochondria, and Psd2p (PtdSer decarboxylase 2 encoded by the PSD2 gene that complements psd2 mutations) in the Golgi. Decarboxylation of PtdSer to PtdEtn (phosphatidylethanolamine) can be used as a biochemical indicator of transport to these organelles, which is regulated by specific lipid and protein motifs. PtdSer transport to mitochondria is controlled by ubiquitination via the action of the ubiquitin ligase subunit Met30p (a ubiquitin ligase subunit encoded by the MET30 gene that complements the met30 mutation affecting methionine biosynthesis). Mutant strains with lesions in the MET30 gene are defective in PtdSer transport and show altered ubiquitination of specific target proteins, such as the transcription factor Met4p (a transcription factor encoded by the MET4 gene that complements the met4 mutation affecting methionine biosynthesis). Mutations to MET30 cause defects in both the MAM as a donor of PtdSer, and the mitochondria as an acceptor of PtdSer in the transport reaction. PtdSer transport to the locus of Psd2p is controlled by specific protein and lipid motifs. The C2 (Ca2+ and phospholipid-binding sequence) domain of Psd2p, and the lipid-binding protein PstB2p (PtdSer transport B pathway protein encoded by the PSTB2 gene that complements the pstB2 mutation affecting PtdSer transport), must be present on acceptor membranes for PtdSer transport to occur. In addition, the action of the PtdIns 4-kinase, Stt4p (PtdIns 4-kinase encoded by the STT4 gene that complements the stt4 mutation causing staurosporine and temperature-sensitive growth) is also required for PtdSer transport to the locus of Psd2p. Reconstitution of PtdSer transport to Psd2p using liposomes demonstrates that PtdSer-rich domains present in vesicles are preferred substrates for transport. In addition, the incorporation of phosphatidic acid into donor membranes enhances the rate of PtdSer transport. Collectively, these data support a model for PtdSer transport in which specific proteins and lipids are required on donor and acceptor membranes.

Protein and lipid motifs regulate phosphatidylserine traffic in yeast

Phosphatidylserine (PtdSer) is synthesized in the endoplasmic reticulum and its subdomains associated with the mitochondria [MAM (mitochondria-associated membrane)] and subsequently transported to the loci of the PtdSer decarboxylases, Pds1p (phosphatidylserine decarboxylase 1 encoded by the PSD1 gene that complements psd1 mutations) in the mitochondria, and Psd2p (PtdSer decarboxylase 2 encoded by the PSD2 gene that complements psd2 mutations) in the Golgi. Decarboxylation of PtdSer to PtdEtn (phosphatidylethanolamine) can be used as a biochemical indicator of transport to these organelles, which is regulated by specific lipid and protein motifs. PtdSer transport to mitochondria is controlled by ubiquitination via the action of the ubiquitin ligase subunit Met30p (a ubiquitin ligase subunit encoded by the MET30 gene that complements the met30 mutation affecting methionine biosynthesis). Mutant strains with lesions in the MET30 gene are defective in PtdSer transport and show altered ubiquitination of specific target proteins, such as the transcription factor Met4p (a transcription factor encoded by the MET4 gene that complements the met4 mutation affecting methionine biosynthesis). Mutations to MET30 cause defects in both the MAM as a donor of PtdSer, and the mitochondria as an acceptor of PtdSer in the transport reaction. PtdSer transport to the locus of Psd2p is controlled by specific protein and lipid motifs. The C2 (Ca2+ and phospholipid-binding sequence) domain of Psd2p, and the lipid-binding protein PstB2p (PtdSer transport B pathway protein encoded by the PSTB2 gene that complements the pstB2 mutation affecting PtdSer transport), must be present on acceptor membranes for PtdSer transport to occur. In addition, the action of the PtdIns 4-kinase, Stt4p (PtdIns 4-kinase encoded by the STT4 gene that complements the stt4 mutation causing staurosporine and temperature-sensitive growth) is also required for PtdSer transport to the locus of Psd2p. Reconstitution of PtdSer transport to Psd2p using liposomes demonstrates that PtdSer-rich domains present in vesicles are preferred substrates for transport. In addition, the incorporation of phosphatidic acid into donor membranes enhances the rate of PtdSer transport. Collectively, these data support a model for PtdSer transport in which specific proteins and lipids are required on donor and acceptor membranes.

RhoGDI-3, a promising system to investigate the regulatory function of rhoGDIs: uncoupling of inhibitory and shuttling functions of rhoGDIs

rhoGDIs (Rho GDP dissociation inhibitors) are postulated to regulate the activity and the localization of small G-proteins of the Rho family by a shuttling process involving extraction of Rho from donor membranes, formation of inhibitory cytosolic rhoGDI/Rho complexes, and delivery of Rho to target membranes. However, the role of rhoGDIs in site-specific membrane targeting or extraction of Rho is still poorly understood. We investigated here the in vivo functions of two mammalian rhoGDIs: the specific rhoGDI-3 and the well-studied rhoGDI-1 (rhoGDI) after structure-based mutagenesis. We identified two sites in rhoGDIs, forming conserved interactions with their Rho target, whose mutation results in the uncoupling of inhibitory and shuttling functions of rhoGDIs in vivo. Remarkably, these rhoGDI mutants were detected at Rho-induced membrane ruffles or protrusions, where they co-localized with RhoG or Cdc42, probably identifying for the first time the site of extraction of a Rho protein by a rhoGDI in vivo. We propose that these mutations act by modifying the steady-state kinetics of the shuttling process regulated by rhoGDIs, such that transient steps at the cell membranes now become detectable. They should provide valuable tools for future investigations of the dynamics of membrane extraction or delivery of Rho proteins and their regulation by cellular partners.

StarD10, a START Domain Protein Overexpressed in Breast Cancer, Functions as a Phospholipid Transfer Protein

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.

Golgi-Localized, γ-Ear-Containing, Arf-Binding Protein Adaptors Mediate Insulin-Responsive Trafficking of Glucose Transporter 4 in 3T3-L1 Adipocytes

Small glucose transporter 4 (Glut4)-containing vesicles represent the major insulin-responsive compartment in fat and skeletal muscle cells. The molecular mechanism of their biogenesis is not yet elucidated. Here, we studied the role of the newly discovered family of monomeric adaptor proteins, GGA (Golgi-localized, gamma-ear-containing, Arf-binding proteins), in the formation of small Glut4 vesicles and acquisition of insulin responsiveness in 3T3-L1 adipocytes. In these cells, all three GGA isoforms are expressed throughout the differentiation process. In particular, GGA2 is primarily present in trans-Golgi network and endosomes where it demonstrates a significant colocalization with the recycling pool of Glut4. Using the techniques of immunoadsorption as well as glutathione-S-transferase pull-down assay we found that Glut4 vesicles (but not Glut4 per se) interact with GGA via the Vps-27, Hrs, and STAM (VHS) domain. Moreover, a dominant negative GGA mutant inhibits formation of Glut4 vesicles in vitro. To study a possible role of GGA in Glut4 traffic in the living cell, we stably expressed a dominant negative GGA mutant in 3T3-L1 adipocytes. Formation of small insulin-responsive Glut4-containing vesicles and insulin-stimulated glucose uptake in these cells were markedly impaired. Thus, GGA adaptors participate in the formation of the insulin-responsive vesicular compartment from the intracellular donor membranes both in vivo and in vitro.

Modulation of Endosomal Cholesteryl Ester Metabolism by Membrane Cholesterol

Cells acquire cholesterol in part by endocytosis of cholesteryl ester containing lipoproteins. In endosomes and lysosomes cholesteryl ester is hydrolyzed by acidic cholesteryl ester hydrolase producing cholesterol and fatty acids. Under certain pathological conditions, however, such as in atherosclerosis, excessive levels of cholesteryl ester accumulate in lysosomes for reasons that are poorly understood. Here, we have studied endosomal and lysosomal cholesteryl ester metabolism in cultured mouse macrophages and with cell-free extracts. We show that net hydrolysis of cholesteryl ester is coupled to the transfer of cholesterol to membranes. When membrane cholesterol levels are low, absorption of cholesterol effectively drives cholesteryl ester hydrolysis. When cholesterol levels in acceptor membranes approach saturation or when cholesterol export is blocked, cholesterol is re-esterified in endosomes. These results reveal a new facet of cellular cholesterol homeostasis and provide a potential explanation for cholesteryl ester accumulation in lysosomes of atherosclerotic cells.

Uncoupling of Inhibitory and Shuttling Functions of Rho GDP Dissociation Inhibitors

Rho GDP dissociation inhibitors (rhoGDIs) are postulated to regulate the activity of small G proteins of the Rho family by a shuttling process involving the extraction of Rho from donor membranes, the formation of the inhibitory cytosolic Rho/rhoGDI complexes, and delivery of Rho to target membranes. However, the role of rhoGDIs in site-specific membrane targeting or extraction of Rho is still poorly understood. Here we investigated the molecular functions of two rhoGDIs, the specific rhoGDI-3 and the less specific but well studied rhoGDI-1, in HeLa cells using structure-based mutagenesis of the rhoGDI protein. We identified two sites in rhoGDI, which form conserved interactions with their Rho target, whose mutation results in the uncoupling of inhibitory and shuttling functions of rhoGDIs: D66GDI-3 (equivalent to D45GDI-1), a conserved residue in the helix-loop-helixGDI/switch 1Rho interface, and D206GDI-3 (equivalent to D185GDI-1) in the beta-sandwichGDI/switch 2Rho interface. Mutations of both sites result in the loss of rhoGDI-3 or rhoGDI-1 inhibitory activity but not of their ability to form cytosolic complexes with RhoG or Cdc42 in vivo. Remarkably, the mutants were detected at Rho-induced membrane ruffles or protrusions where they co-localized with RhoG or Cdc42, likely identifying for the first time the site of extraction of a Rho protein by a rhoGDI in vivo. We propose that these mutations act by modifying the steady-state kinetics of the shuttling process regulated by rhoGDIs, such that transient steps at the cell membranes now become detectable. They should provide valuable tools for future investigations of the dynamics of membrane extraction or delivery of Rho proteins and their regulation by cellular partners.