dGRASP localization and function in the early exocytic pathway in Drosophila S2 cells

The de novo model for Golgi stack biogenesis predicts that membrane exiting the ER at transitional ER (tER) sites contains and recruits all the necessary molecules to form a Golgi stack, including the Golgi matrix proteins, p115, GM130, and GRASP65/55. These proteins leave the tER sites faster than Golgi transmembrane resident enzymes, suggesting that they act as a template nucleating the formation of the Golgi apparatus. However, the localization of the Golgi matrix proteins at tER sites is only shown under conditions where exit from the ER is blocked. Here, we show in Drosophila S2 cells, that dGRASP, the single Drosophila homologue of GRASP65/55, localizes both to the Golgi membranes and the tER sites at steady state and that the myristoylation of glycine 2 is essential for the localization to both compartments. Its depletion for 96 h by RNAi gave an effect on the architecture of the Golgi stacks in 30% of the cells, but a double depletion of dGRASP and dGM130 led to the quantitative conversion of Golgi stacks into clusters of vesicles and tubules, often featuring single cisternae. This disruption of Golgi architecture was not accompanied by the disorganization of tER sites or the inhibition of anterograde transport. This shows that, at least in Drosophila, the structural integrity of the Golgi stacks is not required for efficient transport. Overall, dGRASP exhibits a dynamic association to the membrane of the early exocytic pathway and is involved in Golgi stack architecture.     

Localization of Sed5, a putative vesicle targeting molecule, to the cis- Golgi network involves both its transmembrane and cytoplasmic domains

The yeast Sed5 protein, which is required for vesicular transport between ER and Golgi complex, is a membrane protein of the syntaxin family. These proteins are thought to provide the specific targets that are recognized by transport vesicles. We have investigated the mechanism by which Sed5 protein is itself localized. Expression of epitope-tagged versions of the yeast, Drosophila and rat Sed5 homologues in COS cells results in a perinuclear distribution; immuno- EM reveals that the majority of the protein is in a tubulo-vesicular compartment on the cis side of the Golgi apparatus. A similar distribution was obtained with a chimeric molecule consisting of a plasma membrane syntaxin with the Drosophila Sed5 transmembrane domain. This indicates that the membrane-spanning domain contains targeting information, as is the case with resident Golgi enzymes. However, alterations to the transmembrane domain of Drosophila Sed5 itself did not result in its mistargeting, implying that an additional targeting mechanism exists which involves only the cytoplasmic part of the protein. This was confirmed by modifying the transmembrane domain of the yeast Sed5 protein: substitution with the corresponding region from the Sso1 protein (a plasma membrane syntaxin homologue) did not affect yeast Sed5 function in vivo.     

Signal-anchor sequences are an essential factor for the Golgi-plasma membrane localization of type II membrane proteins

Despite studies of the mechanism underlying the intracellular localization of membrane proteins, the specific mechanisms by which each membrane protein localizes to the endoplasmic reticulum, Golgi apparatus, and plasma membrane in the secretory pathway are unclear. In this study, a discriminant analysis of endoplasmic reticulum, Golgi apparatus and plasma membrane-localized type II membrane proteins was performed using a position-specific scoring matrix derived from the amino acid propensity of the sequences around signal-anchors. The possibility that the sequence around the signal-anchor is a factor for identifying each localization group was evaluated. The discrimination accuracy between the Golgi apparatus and plasma membrane-localized type II membrane proteins was as high as 90%, indicating that, in addition to other factors, the sequence around signal-anchor is an essential component of the selection mechanism for the Golgi and plasma membrane localization. These results may improve the use of membrane proteins for drug delivery and therapeutic applications.     

Apical-basal polarity in Drosophila neuroblasts is independent of vesicular trafficking

The possession of apical-basal polarity is a common feature of epithelia and neural stem cells, so-called neuroblasts (NBs). In Drosophila, an evolutionarily conserved protein complex consisting of atypical protein kinase C and the scaffolding proteins Bazooka/PAR-3 and PAR-6 controls the polarity of both cell types. The components of this complex localize to the apical junctional region of epithelial cells and form an apical crescent in NBs. In epithelia, the PAR proteins interact with the cellular machinery for polarized exocytosis and endocytosis, both of which are essential for the establishment of plasma membrane polarity. In NBs, many cortical proteins show a strongly polarized subcellular localization, but there is little evidence for the existence of distinct apical and basolateral plasma membrane domains, raising the question of whether vesicular trafficking is required for polarization of NBs. We analyzed the polarity of NBs mutant for essential regulators of the main exocytic and endocytic pathways. Surprisingly, we found that none of these mutations affected NB polarity, demonstrating that NB cortical polarity is independent of plasma membrane polarity and that the PAR proteins function in a cell type-specific manner.     

Cell-cell adhesion via the ECM: integrin genetics in fly and worm.

Integrins are essential for the development of the two genetically tractable invertebrate model organisms, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Just two integrins are present in C. elegans: one putative RGD binding integrin alphapat-2betapat-3, corresponding to Drosophila alphaPS2betaPS and vertebrate alpha5beta1, alphaVbeta1 and alpha8beta1, and one putative laminin binding integrin alphaina-1betapat-3, corresponding to Drosophila alphaPS1betaPS and vertebrate alpha3beta1, alpha6beta1 and alpha7beta1. In this review, the function of this minimal set of integrins during the development of these two invertebrates is compared. Despite the differences in bodyplan and developmental strategy, integrin adhesion to the extracellular matrix is required for similar processes: the formation of the link that translates muscle contraction into movement of the exoskeleton, cell migration, and morphogenetic interactions between epithelia. Other integrin functions, such as regulation of gene expression, have not yet been experimentally demonstrated in both organisms. Additional proteins have been characterised in each organism that are essential for integrin function, including extracellular matrix ligands and intracellular interacting proteins, but so far different proteins have been found in the two organisms. This in part represents the fact that the characterisation of the full set of interacting proteins is not complete in either system. However, in other cases different proteins appear to be used for similar functions in the two animals. The continued use of genetic approaches to identify proteins required for integrin function in these two model organisms should lead to the identification of the minimal set of conserved components that form integrin adhesive structures.     

The small G protein Arl1 directs the trans-Golgi-specific targeting of the Arf1 exchange factors BIG1 and BIG2

The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi-specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.     

A region containing a proline-rich motif targets sG(i2) to the golgi apparatus

The central function of heterotrimeric GTP-binding proteins (G proteins) is the transduction of extracellular signals, via membrane receptors, leading to the activation of intracellular effectors. In addition to being associated with the plasma membrane, the alpha subunits of some of these proteins have also been localized in intracellular compartments. The mRNA of the G-protein inhibitory alpha subunit 2 (G(alphai2)) encodes two proteins, G(alphai2) and sG(i2), by an alternative splicing mechanism. sG(i2) differs from G(alphai2) in the C-terminal region and localizes in the Golgi in contrast to the plasma membrane localization of G(alphai2). In this paper we show that the sequence specific to sG(i2) can direct the Golgi localization of other G(alphai) subunits, but not of the stimulatory subunit G(alphas) or of a secreted protein. This indicates that, in addition to the sG(i2) C-terminus, sequences located elsewhere in the protein are required to determine the Golgi localization. Inside the sG(i2) C-terminal region we have identified a 14-amino-acid proline-rich motif which specifies the Golgi localization. Finally, we show that the sG(i2) subunit, once activated, leaves the Golgi to be localized in the endoplasmic reticulum.     

Cholesterol-induced protein sorting: an analysis of energetic feasibility

The mechanism(s) underlying the sorting of integral membrane proteins between the Golgi complex and the plasma membrane remain uncertain because no specific Golgi retention signal has been found. Moreover one can alter a protein's eventual localization simply by altering the length of its transmembrane domain (TMD). M. S. Bretscher and S. Munro (SCIENCE: 261:1280-1281, 1993) therefore proposed a physical sorting mechanism based on the hydrophobic match between the proteins' TMD and the bilayer thickness, in which cholesterol would regulate protein sorting by increasing the lipid bilayer thickness. In this model, Golgi proteins with short TMDs would be excluded from cholesterol-enriched domains (lipid rafts) that are incorporated into transport vesicles destined for the plasma membrane. Although attractive, this model remains unproven. We therefore evaluated the energetic feasibility of a cholesterol-dependent sorting process using the theory of elastic liquid crystal deformations. We show that the distribution of proteins between cholesterol-enriched and cholesterol-poor bilayer domains can be regulated by cholesterol-induced changes in the bilayer physical properties. Changes in bilayer thickness per se, however, have only a modest effect on sorting; the major effect arises because cholesterol changes also the bilayer material properties, which augments the energetic penalty for incorporating short TMDs into cholesterol-enriched domains. We conclude that cholesterol-induced changes in the bilayer physical properties allow for effective and accurate sorting which will be important generally for protein partitioning between different membrane domains.     

Protein trafficking in the exocytic pathway of polarized epithelial cells

Ten years ago, we knew much about the function of polarized epithelia from the work of physiologists, but, as cell biologists, our understanding of how these cells were constructed was poor. We knew proteins were sorted and targeted to different plasma membrane domains and that, in some cells, the Golgi was the site of sorting, but we did not know the mechanisms involved. Between 1991 and the present, significant advances were made in defining sorting motifs for apical and basal-lateral proteins, describing the sorting machinery in the trans-Golgi network (TGN) and plasma membrane, and in understanding how cells specify delivery of transport vesicles to different membrane domains. The challenge now is to extend this knowledge to defining molecular mechanisms in detail in vitro and comprehending the development of complex epithelial structures in vivo.     

Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells

Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.     

The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.

C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines. We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus. Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain. The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane. Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants. Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S. cerevisiae.     

B2-1, a Sec7- and pleckstrin homology domain-containing protein, localizes to the Golgi complex

B2-1 is a human protein that contains both a Sec7 and a pleckstrin homology domain. The yeast Sec7 protein was previously shown to be involved in vesicle formation in the Golgi and endoplasmic reticulum. Recently, several groups have shown that B2-1 and highly similar proteins (e.g., ARNO, ARNO3) have varied cellular functions and subcellular locations. One of these is an association of the B2-1 Sec7 domain with the plasma membrane, binding to the cytoplasmic portion of the integrin beta2 chain (CD18) and is postulated to be involved in inside-out signaling. Other groups have shown that B2-1 and these related proteins are guanine nucleotide-exchange factors that act upon ADP ribosylation factors (ARFs) and are localized to the Golgi or plasma membrane. Here we report the subcellular localization of B2-1 protein. Interestingly, B2-1 does not localize to the plasma membrane but rather associates with a distinct Golgi complex compartment. B2-1's distribution can be disrupted by brefeldin A, a drug that rapidly disrupts the Golgi apparatus by inhibiting ARF activity. Furthermore, transient transfection of GFP-tagged B2-1 shows Golgi complex targeting. Excessive overexpression of transfected B2-1 causes partial Golgi dispersion.     

Exchange factors of the RasGRP family mediate Ras activation in the Golgi

H-Ras and N-Ras become activated both at the plasma membrane and in endomembrane structures such as the Golgi apparatus. This compartmentalized activation is relevant from a signaling standpoint, because effector molecules can become activated differently depending on the region of the cell where Ras proteins are activated. An unsolved question in this new regulatory mechanism is the understanding of how Ras proteins become activated in endomembranes. To approach this problem, we have studied the subcellular distribution and activities of a number of Ras guanosine nucleotide exchange factors. Our results indicate that Ras activation at the plasma membrane and endoplasmic reticulum is an unspecific process that can be achieved by most Ras activators. In contrast, GTP loading of Ras at the Golgi is only induced by members of the Ras guanosine nucleotide releasing protein family. In agreement with these observations, Ras guanosine nucleotide releasing proteins are the only Ras activators showing localization in the Golgi. These results indicate that the compartmentalized activation of effector pathways by Ras proteins depends not only on the specific localization of the GTPases but also in the availability of GDP/GTP exchange factors capable of activating Ras proteins in specific subcellular compartments.     

Golgi maturation‐dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi‐resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra‐Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially‐acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi‐localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra‐Golgi retro‐transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub‐Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.     

Lethal giant larvae take on a life of their own

Cell polarization requires the segregation of the plasma membrane into domains of distinct protein composition. The Lethal giant larvae (Lgl) protein of Drosophila, initially identified as a tumor suppressor, establishes such domains by localizing specific proteins to specific regions of the plasma membrane. However, how it does this remains puzzling and controversial. Recent studies of the yeast orthologs show a molecular pathway through which Lgl is activated locally to promote the targeted fusion of vesicles with the plasma membrane. Here, we reconcile these data with conflicting findings on the mechanism of Lgl in animals and consider if a similar model explains its role in epithelial polarity and asymmetric cell division.     

Caveolin regulates endocytosis of the muscle repair protein, dysferlin

Dysferlin and Caveolin-3 are plasma membrane proteins associated with muscular dystrophy. Patients with mutations in the CAV3 gene show dysferlin mislocalization in muscle cells. By utilizing caveolin-null cells, expression of caveolin mutants, and different mutants of dysferlin, we have dissected the site of action of caveolin with respect to dysferlin trafficking pathways. We now show that Caveolin-1 or -3 can facilitate exit of a dysferlin mutant that accumulates in the Golgi complex of Cav1(-/-) cells. In contrast, wild type dysferlin reaches the plasma membrane but is rapidly endocytosed in Cav1(-/-) cells. We demonstrate that the primary effect of caveolin is to cause surface retention of dysferlin. Caveolin-1 or Caveolin-3, but not specific caveolin mutants, inhibit endocytosis of dysferlin through a clathrin-independent pathway colocalizing with internalized glycosylphosphatidylinositol-anchored proteins. Our results provide new insights into the role of this endocytic pathway in surface remodeling of specific surface components. In addition, they highlight a novel mechanism of action of caveolins relevant to the pathogenic mechanisms underlying caveolin-associated disease.     

Regulation of Integrin β1 Recycling to Lipid Rafts by Rab1a to Promote Cell Migration

Rab1a is a member of the Rab family of small GTPases with a well characterized function in the regulation of vesicle trafficking from the endoplasmic reticulum to the Golgi apparatus and within Golgi compartments. The integrin family heterodimeric transmembrane proteins serve as major receptors for extracellular matrix proteins, which play essential roles in cell adhesion and migration. Although effects on intracellular trafficking of integrins or other key cargos by Rab1a could influence cell migration, the regulatory mechanisms linking Rab1a to cell migration are not well understood. Here, we report identification of Rab1a as a novel regulator of cell migration using an unbiased RNAi screen targeting GTPases. Inhibition of Rab1a reduced integrin-mediated cell adhesion and spreading on fibronectins, reduced integrin β1 localization to lipid rafts, and decreased recycling of integrin β1 to the plasma membrane. Analysis of Rab1a effector molecules showed that p115 mediated Rab1a regulation of integrin recycling and lipid raft localization in cell migration. Taken together, these results suggest a novel function for Rab1a in the regulation of cell migration through controlling integrin β1 recycling and localization to lipid rafts via a specific downstream effector pathway.     

Derlin-dependent retrograde transport from endosomes to the Golgi apparatus

Cells have to maintain stable plasma membrane protein and lipid compositions under normal conditions and to remodel their plasma membranes in response to stimuli. This maintenance and remodeling require that integral membrane proteins at the plasma membrane that become misfolded, because of the relatively harsher extracellular milieu or carbohydrate and amino acid sequence changes, are degraded. We had previously shown that Derlin proteins, required for quality control mechanisms in the endoplasmic reticulum, also localize to endosomes and function in the degradation of misfolded integral membrane proteins at the plasma membrane. In this study, we show that Derlin proteins physically associate with sorting nexins that function in retrograde membrane transport from endosomes to the Golgi apparatus. Using genetic studies in Caenorhabditis elegans and ricin pulse-chase analyses in murine RAW264.7 macrophages, we show that the Derlin-sorting nexin interaction is physiologically relevant. Our studies suggest that at least some integral membrane proteins that are misfolded at the plasma membrane are retrogradely transported to the Golgi apparatus and ultimately to the endoplasmic reticulum for degradation via resident quality control mechanisms.     

Adipsin and the glucose transporter GLUT4 traffic to the cell surface via independent pathways in adipocytes

Insulin increases the exocytosis of many soluble and membrane proteins in adipocytes. This may reflect a general effect of insulin on protein export from the trans Golgi network. To test this hypothesis, we have compared the trafficking of the secreted serine protease adipsin and the integral membrane proteins GLUT4 and transferrin receptors in 3T3-L1 adipocytes. We show that adipsin is secreted from the trans Golgi network to the endosomal system, as ablation of endosomes using transferrin-HRP conjugates strongly inhibited adipsin secretion. Phospholipase D has been implicated in export from the trans Golgi network, and we show that insulin stimulates phospholipase D activity in these cells. Inhibition of phospholipase D action with butan-1-ol blocked adipsin secretion and resulted in accumulation of adipsin in trans Golgi network-derived vesicles. In contrast, butan-1-ol did not affect the insulin-stimulated movement of transferrin receptors to the plasma membrane, whereas this was abrogated following endosome ablation. GLUT4 trafficking to the cell surface does not utilise this pathway, as insulin-stimulated GLUT4 translocation is still observed after endosome ablation or inhibition of phospholipase D activity. Immunolabelling revealed that adipsin and GLUT4 are predominantly localised to distinct intracellular compartments. These data suggest that insulin stimulates the activity of the constitutive secretory pathway in adipocytes possibly by increasing the budding step at the TGN by a phospholipase D-dependent mechanism. This may have relevance for the secretion of other soluble molecules from these cells. This is not the pathway employed to deliver GLUT4 to the plasma membrane, arguing that insulin stimulates multiple pathways to the cell surface in adipocytes.     

H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway

Ras proteins must be localized to the inner surface of the plasma membrane to be biologically active. The motifs that effect Ras plasma membrane targeting consist of a C-terminal CAAX motif plus a second signal comprising palmitoylation of adjacent cysteine residues or the presence of a polybasic domain. In this study, we examined how Ras proteins access the cell surface after processing of the CAAX motif is completed in the endoplasmic reticulum (ER). We show that palmitoylated CAAX proteins, in addition to being localized at the plasma membrane, are found throughout the exocytic pathway and accumulate in the Golgi region when cells are incubated at 15 degrees C. In contrast, polybasic CAAX proteins are found only at the cell surface and not in the exocytic pathway. CAAX proteins which lack a second signal for plasma membrane targeting accumulate in the ER and Golgi. Brefeldin A (BFA) significantly inhibits the plasma membrane accumulation of newly synthesized, palmitoylated CAAX proteins without inhibiting their palmitoylation. BFA has no effect on the trafficking of polybasic CAAX proteins. We conclude that H-ras and K-ras traffic to the cell surface through different routes and that the polybasic domain is a sorting signal diverting K-Ras out of the classical exocytic pathway proximal to the Golgi. Farnesylated Ras proteins that lack a polybasic domain reach the Golgi but require palmitoylation in order to traffic further to the cell surface. These data also indicate that a Ras palmitoyltransferase is present in an early compartment of the exocytic pathway.