Role In Membrane Trafficking Research Articles

Rab11b Is Essential for Recycling of Transferrin to the Plasma Membrane

Members of the Rab family of small GTPases play important roles in membrane trafficking along the exocytic and endocytic pathways. The Rab11 subfamily consists of two highly conserved members, Rab11a and Rab11b. Rab11a has been localized both to the pericentriolar recycling endosome and to the trans-Golgi network and functions in recycling of transferrin. However, the localization and function of Rab11b are completely unknown. In this study green fluorescent protein (GFP)-tagged Rab11b was used to determine its subcellular localization. GFP-Rab11b colocalized with internalized transferrin, and using different mutants of Rab11b, the role of this protein in transferrin uptake and recycling was examined. Two of these mutants, Rab11b-Q/L (constitutively active) and Rab11b-S/N (constitutively inactive), strongly inhibited the recycling of transferrin. Interestingly, both of them had no effect on transferrin uptake. In contrast, the C-terminally altered mutant Rab11b-ΔC, which cannot be prenylated and therefore cannot interact with membranes, did not interfere with wild-type Rab11b function. From these data we concluded that functional Rab11b is essential for the transport of internalized transferrin from the recycling compartment to the plasma membrane.

SYNCRIP, a Cytoplasmic Counterpart of Heterogeneous Nuclear Ribonucleoprotein R, Interacts with Ubiquitous Synaptotagmin Isoforms

Synaptotagmins (Syts) are a large family of membrane proteins consisted of at least 12 isoforms. They are categorized in neuron-specific isoforms (I-V, X, and XI) and ubiquitous isoforms (VI-IX) based on their expression patterns. Syt-I, a neuron-specific and abundant isoform, has been well characterized and postulated to be the exocytotic Ca(2+) sensor. However, the functions of other isoforms remain obscure. Here, we report that ubiquitous isoforms of synaptotagmins, Syt-VII, Syt-VIII, and Syt-IX, interacted with a cytoplasmic RNA-binding protein, SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein), through their C2B domains. SYNCRIP was originally found in the Syt-II C2AB domain bound fraction from the mouse brain lysate. cDNA cloning of SYNCRIP cDNA revealed that the protein was highly homologous to heterogeneous nuclear ribonucleoprotein R (hnRNP R) recently identified. SYNCRIP protein was ubiquitously and constantly expressed in various tissues of mice parallel to hnRNP R. SYNCRIP indeed bound RNA with preference to poly(A) RNA; however, in contrast to the nuclear localization of hnRNP R, SYNCRIP was distributed predominantly in the cytoplasm as judged by both biochemical fractionation and immunohistochemical studies. In vitro binding experiments showed the potential interaction of SYNCRIP with C2B domains of Syts except for those of Syt-V, -VI, and -X. Furthermore, the interaction between SYNCRIP and Syt-VII, -VIII, or -IX was revealed by co-immunoprecipitation experiments using COS cells transiently expressing each Syt isoform. These findings suggested that SYNCRIP was a target of ubiquitous type of Syts and implied the involvement of ubiquitous Syts in the regulation of dynamics of the cytoplasmic mRNA.

Syntaxin 1A inhibits regulated CFTR trafficking in xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial cell Cl channel, whose gating activity and membrane trafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett, D. J. Benos, M. W. Quick, and K. L. Kirk. Nature 390: 302-305, 1997), a protein best known for its role in membrane trafficking and neurosecretion. To examine the mechanism of syntaxin 1A inhibition, we expressed these proteins in Xenopus oocytes and monitored agonist-induced changes in plasma membrane capacitance and cell surface fluorescence of CFTR that contains an external epitope tag. cAMP stimulation elicited large increases in membrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTR content. Injection of botulinum toxin/C1 rapidly reversed syntaxin's effects on current and capacitance, indicating that they cannot be explained by an effect on CFTR synthesis. Functional expression of other integral membrane proteins, including Na-coupled glucose transporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotide receptor, and viral hemagglutinin protein, was not affected by syntaxin 1A coexpression. These findings indicate that acute regulation of the number of CFTR Cl channels in plasma membrane is one mechanism by which cAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTR content by syntaxin 1A is consistent with the concept that syntaxin and other components of the SNARE machinery are involved in regulated trafficking of CFTR.

Crystal Structure of the Vesicular Transport Protein Sec17: Implications for SNAP Function in SNARE Complex Disassembly

SNAP proteins play an essential role in membrane trafficking in eukaryotic cells. They activate and recycle SNARE proteins by serving as adaptors between SNAREs and the cytosolic chaperone NSF. We have determined the crystal structure of Sec17, the yeast homolog of α-SNAP, to 2.9 Å resolution. Sec17 is composed of an N-terminal twisted sheet of α-helical hairpins and a C-terminal α-helical bundle. The N-terminal sheet has local similarity to the tetratricopeptide repeats from protein phosphatase 5 but has a different overall twist. Sec17 also shares structural features with HEAT and clathrin heavy chain repeats. Possible models of SNAP:SNARE binding suggest that SNAPs may function as lever arms, transmitting forces generated by conformational changes in NSF/Sec18 to drive disassembly of SNARE complexes.

SNARE membrane trafficking dynamics in vivo.

The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in approximately 1-micrometer cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at approximately 1-micrometer ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched approximately 1-micrometer peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.

Rapid arrest of axon elongation by brefeldin A: a role for the small GTP-binding protein ARF in neuronal growth cones.

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.

Three Novel Proteins of the Syntaxin/SNAP-25 Family

Intracellular membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) through the formation of complexes between these proteins present on vesicle and target membranes. All known SNARE-mediated fusion events involve members of the syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of a large number of different syntaxin and vesicle-associated membrane protein genes. To further investigate the spectrum of SNAREs and their roles in membrane trafficking we characterized three novel members of the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) subfamilies. The proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple membranes. SNAP-29 does not contain a predicted membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of syntaxins.

Presenilins, the endoplasmic reticulum, and neuronal apoptosis in Alzheimer's disease.

Many cases of autosomal dominant inherited forms of early-onset Alzheimer's disease are caused by mutations in the genes encoding presenilin-1 (PS-1; chromosome 14) and presenilin-2 (PS-2; chromosome 1). PSs are expressed in neurons throughout the brain wherein they appear to be localized primarily to the endoplasmic reticulum (ER) of cell bodies and dendrites. PS-1 and PS-2 show high homology and are predicted to have eight transmembrane domains with the C terminus, N terminus, and a loop domain all on the cytosolic side of the membrane; an enzymatic cleavage of PSs occurs at a site near the loop domain. The normal function of PSs is unknown, but data suggest roles in membrane trafficking, amyloid precursor protein processing, and regulation of ER calcium homeostasis. Homology of PSs to the C. elegans gene sel-12, which is involved in Notch signaling, and phenotypic similarities of PS-1 and Notch knockout mice suggest a developmental role for PSs in the nervous system. When expressed in cultured cells and transgenic mice, mutant PSs promote increased production of a long form of amyloid beta-peptide (A beta1-42) that may possess enhanced amyloidogenic and neurotoxic properties. PS mutations sensitize cultured neural cells to apoptosis induced by trophic factor withdrawal, metabolic insults, and amyloid beta-peptide. The mechanism responsible for the proapoptotic action of mutant PSs may involve perturbed calcium release from ER stores and increased levels of oxidative stress. Recent studies of apoptosis in many different cell types suggest that ER calcium signaling can modulate apoptosis. The evolving picture of PS roles in neuronal plasticity and Alzheimer's disease is bringing to the forefront the ER, an organelle increasingly recognized as a key regulator of neuronal plasticity and survival.

Myosin VIIA Is Required for Aminoglycoside Accumulation in Cochlear Hair Cells

Myosin VIIA is expressed by sensory hair cells and has a primary structure predicting a role in membrane trafficking and turnover, processes that may underlie the susceptibility of hair cells to aminoglycoside antibiotics. [3H]Gentamicin accumulation and the effects of aminoglycosides were therefore examined in cochlear cultures of mice with different missense mutations in the myosin VIIA gene, Myo7a, to see whether myosin VIIA plays a role in aminoglycoside ototoxicity. Hair cells from homozygous mutant Myo7ash1 mice, with a mutation in a nonconserved region of the myosin VIIA head, respond rapidly to aminoglycoside treatment and accumulate high levels of gentamicin. Hair cells from homozygous mutant Myo7a6J mice, with a mutation at a highly conserved residue close to the ATP binding site of the myosin VIIA head, do not accumulate [3H]gentamicin and are protected from aminoglycoside ototoxicity. Hair cells from heterozygotes of both alleles accumulate [3H]gentamicin and respond to aminoglycosides. Although aminoglycoside uptake is thought to be via apical surface-associated endocytosis, coated pit numbers on the apical membrane of heterozygous and homozygous Myo7a6J hair cells are similar. Pulse-chase experiments with cationic ferritin confirm that the apical endocytotic pathway is functional in homozygous Myo7a6J hair cells. Transduction currents can be recorded from both heterozygous and homozygous Myo7a6J hair cells, suggesting it is unlikely that the drug enters via diffusion through the mechanotransducer channel. The results show that myosin VIIA is required for aminoglycoside accumulation in hair cells. Myosin VIIA may transport a putative aminoglycoside receptor to the hair cell surface, indirectly translocate it to sites of membrane retrieval, or retain it in the endocytotic pathway.

Cell and molecular neurobiology of presenilins: a role for the endoplasmic reticulum in the pathogenesis of Alzheimer's disease?

Mutations in genes encoding presenilin-1 (PS-1) and presenilin-2 (PS-2) cause many cases of autosomal dominant inherited forms of early-onset Alzheimer's disease (AD). PSs are expressed in neurons throughout the nervous system, with differences in abundance among cell populations. PS-1 and PS-2 each have six to eight transmembrane domains and are localized mainly in the endoplasmic reticulum (ER). PSs may interact with cytoskeletal proteins and beta-amyloid precursor protein (APP) in ways consistent with roles in membrane trafficking and APP processing. Expression of mutant PSs in cultured cells and transgenic mice results in increased production of an amyloidogenic-cytotoxic form of amyloid beta-peptide (Abeta). Neural cells expressing mutant PSs exhibit increased sensitivity to apoptosis induced by trophic factor withdrawal and Abeta. The proapoptotic action of mutant PSs involves perturbed calcium release from ER stores and increased levels of oxidative stress. PS mutations may also suppress neurotransmitter synthesis in cholinergic neurons, suggesting a role in regulation of neuronal phenotype. Homology of PSs with the C. elegans gene sel-12 and phenotypic similarities of PS-1 and Notch knockout mice suggest a developmental role for PSs in somitogenesis. Collectively, the emerging data suggest intriguing roles of PSs in neuronal plasticity and cell death and highlight the importance of the ER as a regulatory site involved in the pathogenesis of neuronal degeneration in AD.

Role of membrane trafficking in plasma membrane solute transport.

Cells can rapidly and reversibly alter solute transport rates by changing the kinetics of transport proteins resident within the plasma membrane. Most notably, this can be brought about by reversible phosphorylation of the transporter. An additional mechanism for acute regulation of plasma membrane transport rates is by the regulated exocytic insertion of transport proteins from intracellular vesicles into the plasma membrane and their subsequent regulated endocytic retrieval. Over the past few years, the number of transporters undergoing this regulated trafficking has increased dramatically, such that what was once an interesting translocation of a few transporters has now become a widespread modality for regulating plasma membrane solute permeabilities. The aim of this article is to review the models proposed for the regulated trafficking of transport proteins and what lines of evidence should be obtained to document regulated exocytic insertion and endocytic retrieval of transport proteins. We highlight four transporters, the insulin-responsive glucose transporter, the antidiuretic hormone-responsive water channel, the urinary bladder H(+)-ATPase, and the cystic fibrosis transmembrane conductance regulator Cl- channel, and discuss the various approaches taken to document their regulated trafficking. Finally, we discuss areas of uncertainty that remain to be investigated concerning the molecular mechanisms involved in regulating the trafficking of proteins.

Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor-containing vesicles in CHO cells.

Cellubrevin is a member of the synaptobrevin/VAMP family of SNAREs, which has a broad tissue distribution. In fibroblastic cells it is concentrated in the vesicles which recycle transferrin receptors but its role in membrane trafficking and fusion remains to be demonstrated. Cellubrevin, like the synaptic vesicle proteins synaptobrevins I and II, can be cleaved by tetanus toxin, a metallo-endoprotease which blocks neurotransmitter release. However, nonneuronal cells are unaffected by the toxin due to lack of cell surface receptors for its heavy chain. To determine whether cellubrevin cleavage impairs exocytosis of recycling vesicles, we tested the effect of tetanus toxin light chain on the release of preinternalized transferrin from streptolysin-O-perforated CHO cells. The release was found to be temperature and ATP dependent as well as NEM sensitive. Addition of tetanus toxin light chain, but not of a proteolytically inactive form of the toxin, resulted in a partial inhibition of transferrin release which correlated with the toxin-mediated cleavage of cellubrevin. The residual release of transferrin occurring after complete cellubrevin degradation was still ATP dependent. Our results indicate that cellubrevin plays an important role in the constitutive exocytosis of vesicles which recycle plasmalemma receptors. The incomplete inhibition of transferrin release produced by the toxin suggests the existence of a cellubrevin-independent exocytotic mechanism, which may involve tetanus toxin-insensitive proteins of the synaptobrevin/VAMP family.

Comparison of human normal and neoplastic prostate epithelial cells by TEM and quantitative image analysis and morphometry (QIAM)

Prostate cancer is the most common tumor in men in the U.S.A. The regulation of growth control in normal prostate cells and its contribution to tumor progression is a subject of intense investigation.Recently our group developed culture techniques that allowed us to study normal and neoplastic human prostate epithelial cells from human prostatectomy specimens. Since quantitative evaluation of intracellular compartments will be critical to defining the role of membrane trafficking in the growth regulation, we used TEM and QIAM to characterize normal and neoplastic prostate epithelial cells invivo, as well as in vitro. This work will serve as a baseline to subsequent studies using perturbants of cell trafficking and conditions ;hat promote or block cellular growth, to investigate the role of growth factors and stromal/epithelial nteraction in prostate growth regulation.Normal and neoplastic tissues were sampled for ultrastructural examination from grid mapped radical prostatectomy specimens that were processed for whole mounts. The SV40 immortalized "normal" prostate cell line (P69) was provided by Dr. Strom.

Differential expression of transcripts from syb, a Drosophila melanogaster gene encoding VAMP (synaptobrevin) that is abundant in non-neuronal cells

VAMP (synaptobrevin) is a highly conserved membrane protein originally described as a component of brain synaptic vesicles. The Drosophila melanogaster VAMP-encoding gene (syb) comprises five exons. Splicing exons 1,2,3,4,5 ( syb-b) results in a protein with a C-terminal hydrophobic domain and a negligible intraluminal domain. Splicing exons 1,2,3,5 ( syb-a) predicts a protein with a 20-amino-acid luminal domain at the C terminus. The ratio of syb-a to syb-b transcripts is highly regulated during development. The syb transcripts show no enrichment in the nervous system and are present in very early embryos, well before neurogenesis. The greatest concentration of syb transcripts was found in cells of the gut and malpighian tubules. Thus, syb may have a general role in membrane trafficking and, perhaps, a role in the secretion of digestive enzymes.

Expression of smg p25A/ rab 3A guanine nucleotide dissociation inhibitor (GDI) in neurons and glial cells from rat brain

smg p25A/ rab 3A is a member of the rab family of small guanosine 5′-trisphosphate (GTP)-binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. In neural tissues, smg p25A/ rab 3A is expressed only in neurons but not in glia. In this study, expression of a regulatory protein for smg p25A/ rab 3A, guanine nucleotide dissociation inhibitor ( smg p25A/ rab 3A GDI), in neurons and glia isolated from rat brain was examined. The smg p25A/ rab 3A GDI mRNA was detected as early as on gestational day 14 in the fetal brain, and its level reached a maximum 5 days after birth. By immunoblot analysis, smg p25A/ rab 3A GDI was detected in the homogenate of rat hippocampus as well as in that of astrocytes in primary culture. By immunocytochemical analysis, the smg p25A/ rab 3A GDI immunoreactivity was detected in hippocampal neurons and oligodendrocytes. The immunoreactivity of astrocytes was much lower than that of neurons and oligodendrocytes. These results indicate that smg p25A/ rab 3A GDI may play a fundamental role in membrane trafficking both neurons and glia by interacting with multiple rab family proteins.

Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference to the fractions of these proteins which are membrane-bound. In addition to EGTA-extractable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilized with detergents (Triton X-100 or Triton X-114). A strong base like Na 2CO 3, which is usually effective in extracting peripheral membrane proteins, only partially solubilizes the membrane-bound, EGTA-resistant annexins analyzed here. Also, only 50–60% of the Triton X-114-soluble annexins partition in the aqueous phase, the remaining fractions being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as yet unknown mechanism, following Ca 2+-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of these proteins remain bound to membranes in a Ca 2+-independent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.

The cortical actin-membrane cytoskeleton of unfertilized sea urchin eggs: Analysis of the spatial organization and relationship of filamentous actin, nonfilamentous actin, and egg spectrin

Whole mounts, cryosections, and isolated cortices of unfertilized sea urchin eggs were probed with fluorescent phalloidin, anti-actin and anti-egg spectrin antibodies to investigate the organizational state of the cortically associated actin-membrane cytoskeleton. Filamentous actin and egg spectrin were localized to the plasma membrane, within microvillar and nonmicrovillar domains. The nonmicrovillar filamentous actin was located immediately subjacent to the microvilli forming an extensive interconnecting network along the inner surface of the plasma membrane. The organization of this filamentous actin network precisely correlated with the positioning of the underlying cortical granules. The cortical cytoplasm did not contain any detectable filamentous actin, but instead contained a sequestered domain of nonfilamentous actin. Spectrin was localized to the cytoplasmic surface of the plasma membrane with concentrated foci co-localized with the filamentous actin present in microvilli. Spectrin was also observed to coat the surfaces of cortical granules as well as other populations of intracellular vesicles. On the basis of light microscopic morphology, intracellular distribution, and co-isolation with the egg cortex, some of these spectrin-coated organelles represent acidic vesicles. Identification of an elaborate organization of inter-related domains of actin (filamentous and nonfilamentous) and spectrin forming the cortical membrane cytoskeleton provides insight into the fundamental mechanisms for early membrane restructuring during embryogenesis. Additionally, the localization of spectrin to the surface of intracellular vesicles is indicative of its newly identified functional roles in membrane trafficking, membrane biogenesis and cellular differentiation.

The site of diphosphoinositide synthesis in rat liver

The four mammalian phosphatidylinositol 4-kinases modulate inter-organelle lipid trafficking, phosphoinositide signalling and intracellular vesicle trafficking. In addition to catalytic domains required for the synthesis of PI4P, the phosphatidylinositol 4-kinases also contain isoform-specific structural motifs that mediate interactions with proteins such as AP-3 and the E3 ubiquitin ligase Itch, and such structural differences determine isoform-specific roles in membrane trafficking. Moreover, different permutations of phosphatidylinositol 4-kinase isozymes may be required for a single cellular function such as occurs during distinct stages of GPCR signalling and in Golgi to lysosome trafficking. Phosphatidylinositol 4-kinases have recently been implicated in human disease. Emerging paradigms include increased phosphatidylinositol 4-kinase expression in some cancers, impaired functioning associated with neurological pathologies, the subversion of PI4P trafficking functions in bacterial infection and the activation of lipid kinase activity in viral disease. We discuss how the diverse and sometimes overlapping functions of the phosphatidylinositol 4-kinases present challenges for the design of isoform-specific inhibitors in a therapeutic context.