The Hsp90 chaperone complex regulates GDI-dependent Rab recycling

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.     

Ubiquitously expressed GPCR membrane-trafficking orthologs

Olfactory receptors are a diverse set of G-protein-coupled receptors (GPCRs) that localize to cellular plasma membranes in the olfactory epithelium. Associated trafficking proteins often assist in targeting these GPCRs to the membrane, facilitating function. One such trafficking protein has been isolated as a mutant defective for both odorant response and proper receptor localization in Caenorhabditis elegans. This gene (ODR-4) allows for functional expression of olfactory receptors in heterologous cells that are otherwise incapable of targeting. We have isolated a full-length human cDNA that is homologous to the C. elegans gene at the protein level across nearly the entire gene by using a novel RecA-based gene enrichment procedure. This sequence is homologous to a family of orthologs that share predicted structural features, indicating a conserved function. The gene was expressed in 41 of 44 human, mouse, and rat tissues, suggesting an important role in trafficking olfactory and other GPCRs.     

A Highlights from MBoC Selection: Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.     

Normal Bone Deposition Occurs in Mice Deficient in Factor XIII-A and Transglutaminase 2

Transglutaminase activity has been widely implicated in bone deposition. A predominant role has been proposed for factor (F)XIII-A and a subsidiary role suggested for the homologous protein, transglutaminase 2. Full-length FXIII-A is an 83 kDa protransglutaminase that is present both in plasma and also in haematopoietic and connective tissue lineages. Several studies have reported expression in murine cells, including osteocytes, of a 37 kDa protein that reacts with the monoclonal anti-FXIII-A antibody AC-1A1. This protein was presumed to be a catalytically active fragment of FXIII-A-83 and to play a major role in bone deposition. We detected a 37 kDa AC-1A1 reactive protein in FXIII-A mRNA negative cell lines and in tissues from FXIII-A^−/− mice. By mass spectrometric sequencing of AC-1A1 immunoprecipitates, we identified this protein as transaldolase-1, and confirmed that recombinant transaldolase-1 is recognised by AC-1A1. We have also shown that bone deposition is normal in FXIII-A^−/−.TG2^−/− double knockout mice, casting doubt on the role of transglutaminases in bone mineralisation. Various studies have used antibody AC-1A1 for immunohistochemistry or immunofluorescence. We observe strong FXIII-A dependent staining in paraffin embedded mouse heart sections, with relatively low background in non-expressing mouse cells. In contrast, FXIII-A independent staining predominates in cultured human cells using a standard immunofluorescence procedure. Immunofluorescence is present in membrane compartments that are expected to lack transaldolase, indicating that other off-target antigens are recognised by AC-1A1. This has significant implications for studies that have used this approach to define the subcellular trafficking of FXIII-A in osteocytes.     

Palmitoylation of LAT contributes to its subcellular localization and stability

Palmitoylation is a protein modification for trafficking to lipid raft. Without palmitoylation, linker for activation of T cells (LAT), an adaptor molecule mediating T cell receptor signaling, is unable to localize in lipid rafts and to mediate T cell activation. We here show a novel role for palmitoylation in LAT trafficking to the plasma membrane and in the stability of the LAT protein. The human LAT mutant lacking palmitoylation was unable to traffic to the plasma membrane despite the presence of transmembrane portion. The mouse LAT mutant lacking palmitoylation was unstable and susceptible to degradation via the proteasome pathway. The human LAT mutant became unstable when the extracellular portion was swapped for that from mouse, indicating that both palmitoylation and the extracellular portion regulate the stability of LAT. These results suggest that palmitoylation has an important role in trafficking to the plasma membrane and the stability of LAT.     

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles.Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.     

Isolation and characterization of Golgi apparatus-specific GODZ with the DHHC zinc finger domain

We identified a novel Golgi apparatus-specific protein with the DHHC zinc finger domain and four putative transmembrane regions, designated as GODZ. The amino acid sequences were highly conserved among mouse and human GODZs and homologous proteins in human, mouse, rat, Drosophila melanogaster, and Caenorhabditis elegans, implying a functional significance of the GODZ protein family. Overexpression of mouse GODZ in COS7 cells suppressed the sorting of the glutamate receptor GluRalpha1 from the Golgi apparatus. These results suggest that GODZ plays a role in the membrane protein trafficking.     

Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression

Most cells in tissues are polarized and usually have two distinct plasma membrane domains-an apical membrane and a basolateral membrane, which are the result of polarized trafficking of proteins and lipids. However, the mechanism underlying the cell polarization is not fully understood. In this study, we investigated the involvement of synaptotagmin-like protein 2-a (Slp2-a), an effector molecule for the small GTPase Rab27, in polarized trafficking by using Madin-Darby canine kidney II cells as a model of polarized cells. The results show that the level of Slp2-a expression in MDCK II cells increases greatly as the cells become polarized and that its expression is specifically localized at the apical membrane. The results also reveal that Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in a Rab27A-dependent manner. In addition, ezrin, a downstream target of podocalyxin, and ERK1/2 are activated in Slp2-a-knockdown cells, and their activation results in a dramatic reduction in the amount of the tight junction protein claudin-2. Because both Slp2-a and claudin-2 are highly expressed in mouse renal proximal tubules, Slp2-a is likely to regulate claudin-2 expression through trafficking of podocalyxin to the apical surface in mouse renal tubule epithelial cells.     

Unveiling ubiquitinome rearrangements induced by Salmonella infection

Ubiquitination plays a critical role in the activation of host immune responses to infection and serves as a signal for pathogen delivery to phagophores along the xenophagy pathway. We recently performed systematic ubiquitination site profiling of epithelial cells infected with Salmonella Typhimurium. Our findings specifically highlight components of the NFKB, membrane trafficking pathways and RHO GTPase systems as ubiquitination hubs during infection. In addition, a broad spectrum of bacterial effectors and several outer membrane proteins are ubiquitinated in infected cells. This comprehensive resource of ubiquitinome dynamics during Salmonella infection enables further understanding of the complex host-pathogen interplay and may reveal novel targets for the inhibition of Salmonella invasion and inflammation.     

Sweet delivery - sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells

Antisense oligodeoxynucleotides (ODNs) are short (12-25 nt long) stretches of single-stranded DNA that may be delivered to a cell, where they hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. Here we used confocal microscopy to monitor the uptake and trafficking of ODNs in barley tissues. We conclude that uptake of ODNs across the plant plasma membrane is mediated by active transport of mono- or disaccharides through sugar translocators. We demonstrate that sugar transport can deliver ODNs to barley seeds, and that this strategy may be employed to suppress gene activity in endosperm cells by antisense ODN inhibition. We further found that sucrose compared favorably with oligofectamine as a vehicle for ODN delivery to human cells in a low-serum environment.     

Coordinated protein sorting, targeting and distribution in polarized cells

The polarized distribution of functions in polarized cells requires the coordinated interaction of three machineries that modify the basic mechanisms of intracellular protein trafficking and distribution. First, intrinsic protein-sorting signals and cellular decoding machineries regulate protein trafficking to plasma membrane domains; second, intracellular signalling complexes define the plasma membrane domains to which proteins are delivered; and third, proteins that are involved in cell-cell and cell-substrate adhesion orientate the three-dimensional distribution of intracellular signalling complexes and, accordingly, the direction of membrane traffic. The integration of these mechanisms into a complex and dynamic network is crucial for normal tissue function and is often defective in disease states.     

Distinct Human and Mouse Membrane Trafficking Systems for Sweet Taste Receptors T1r2 and T1r3

The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.     

The lysosomal trafficking regulator interacting protein-5 localizes mainly in epithelial cells

Endocytosis, subsequent protein sorting into multivesicular bodies (MVBs), and eventual degradation in lysosomes compose an important mechanism for controlling protein expression on the plasma membrane. The lysosomal trafficking regulator interacting protein-5 (LIP5) is part of the complex protein machinery involved in MVB biosynthesis. LIP5 interacts with other players of the ESCRT machinery as well as with two known cargo proteins, AQP2 and EGFR, whose degradation is affected upon reduction of LIP5 expression. To investigate the expression and localization pattern of LIP5, we studied LIP5 protein expression in a mouse tissue panel and subjected various rodent and human tissues to immunohistochemistry. Immunoblotting revealed that, except for jejunum, LIP5 is expressed as a 42 kDa protein in all mouse tissues tested. Alternatively-spliced gene products could not be detected. Immunohistochemical studies revealed that in tissues positive for LIP5, LIP5 is detected in virtually all epithelial cells of the examined rodent and human tissues. The observed LIP5 expression in epithelial tissues suggests that LIP5 is of particular importance in the MVB sorting and degradation of proteins expressed in polarized cells.     

Use of colloidal gold cytochemistry in the study of the basic cell biology of cancer

We are currently investigating the morphologic aspects of two areas of the basic cell biology of cancer: tumor-specific surface antigens as targets for immunotoxins, and the phenomenon of multidrug resistance in chemotherapy of human tumors. Colloidal gold cytochemistry has provided a useful method for the electron-microscopic cytochemical detection of materials endocytosed by cells in culture. This technique has been used to study the internalization pathway of ligands bound to the surface of cancer cells, particularly antibodies for use as immunologic targeting reagents for the construction of immunotoxins. These colloidal gold conjugates with monoclonal antibodies have demonstrated the internalization of these immunologic reagents through coated pits and receptosomes, which is a necessary step in the delivery of immunotoxins into the cell where they can mediate their cell-killing functions. Morphologic methods have been employed for the screening and selection of monoclonal antibodies reactive with the surface of human ovarian cancer cells for use as immunotoxins and have demonstrated the in vivo activity of immunotoxins made with these antibodies and Pseudomonas exotoxin in a nude mouse model system. In other studies, we have employed such reagents for the immunocytochemical detection of the surface expression of P170, the cell-surface efflux pump protein responsible for the phenotype of multidrug resistance in tumor cells, and to investigate the distribution of this protein by using immunocytochemistry in normal human tissues. These results have suggested a role for P170 in normal cell membrane transport of metabolites in various organ systems.     

Subcellular localization and targeting of N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the second enzyme in the glycosylphosphatidylinositol biosynthetic pathway

The second step in glycosylphosphatidylinositol biosynthesis is the de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) catalyzed by N-acetylglucosaminylphosphatidylinositol deacetylase (PIG-L). Previous studies of mouse thymoma cells showed that GlcNAc-PI de-N-acetylase activity is localized to the endoplasmic reticulum (ER) but enriched in a mitochondria-associated ER membrane (MAM) domain. Because PIG-L has no readily identifiable ER sorting determinants, we were interested in learning how PIG-L is localized to the ER and possibly enriched in MAM. We used HeLa cells transiently or stably expressing epitope-tagged PIG-L variants or chimeric constructs composed of elements of PIG-L fused to Tac antigen, a cell surface protein. We first analyzed the subcellular distribution of PIG-L and Glc-NAc-PI-de-N-acetylase activity and then studied the localization of Tac-PIG-L chimeras to identify sequence elements in PIG-L responsible for its subcellular localization. We show that human PIG-L is a type I membrane protein with a large cytoplasmic domain and that, unlike the result with mouse thymoma cells, both PIG-L and GlcNAc-PI-de-N-acetylase activity are uniformly distributed between ER and MAM in HeLa cells. Analyses of a series of Tac-PIG-L chimeras indicated that PIG-L contains two ER localization signals, an independent retention signal located between residues 60 and 88 of its cytoplasmic domain and another weak signal in the luminal and transmembrane domains that functions autonomously in the presence of membrane proximal residues of the cytoplasmic domain that themselves lack any retention information. We conclude that PIG-L, like a number of other ER membrane proteins, is retained in the ER through a multi-component localization signal rather than a discrete sorting motif.     

Evolutionary implications of localization of the signaling scaffold protein Parafusin to both cilia and the nucleus

Parafusin (PFUS), a 63 kDa protein first discovered in the eukaryote Paramecium and known for its role in apicomplexan exocytosis, provides a model for the common origin of cellular systems employing scaffold proteins for targeting and signaling. PFUS is closely related to eubacterial rather than archeal phosphoglucomutases (PGM) – as we proved by comparison of their 88 sequences – but has no PGM activity. Immunofluorescence microscopy analysis with a PFUS‐specific peptide antibody showed presence of this protein around the base region of primary cilia in a variety of mammalian cell types, including mouse embryonic (MEFs) and human foreskin fibroblasts (hFFs), human carcinoma stem cells (NT‐2 cells), and human retinal pigment epithelial (RPE) cells. Further, PFUS localized to the nucleus of fibroblasts, and prominently to nucleoli of MEFs. Localization studies were confirmed by Western blot analysis, showing that the PFUS antibody specifically recognizes a single protein of ca. 63 kDa in both cytoplasmic and nuclear fractions. Finally, immunofluorescence microscopy analysis showed that PFUS localized to nuclei and cilia in Paramecium. These results support the suggestion that PFUS plays a role in signaling between nucleus and cilia, and that the cilium and the nucleus both evolved around the time of eukaryotic emergence. We hypothesize that near the beginnings of eukaryotic cell evolution, scaffold proteins such as PFUS arose as peripheral membrane protein identifiers for cytoplasmic membrane trafficking and were employed similarly during the subsequent evolution of exocytic, nuclear transport, and ciliogenic mechanisms.