Regulation of Salmonella-induced neutrophil transmigration by epithelial ADP-ribosylation factor 6.

Salmonella typhimurium elicits an acute inflammatory response in the host intestinal epithelium, characterized by the movement of polymorphonuclear leukocytes (PMN) across the epithelial monolayer to the intestinal lumen. It was recently shown that SipA, a protein secreted by S. typhimurium, is necessary and sufficient to drive PMN transmigration across model intestinal epithelia (Lee, C. A., Silva, M., Siber, A. M., Kelly, A. J., Galyov, E., and McCormick, B. A. (2000) Proc. Natl. Acad Sci. USA 97, 12283-12288). However, the epithelial factors responsible for this process have not been identified. Here, for the first time, we demonstrate that S. typhimurium-induced PMN transmigration across Madin-Darby canine kidney-polarized monolayers is regulated by the GTPase ARF6. Apically added S. typhimurium promoted the translocation of ARF6 and its exchange factor ARNO to the apical surface. Overexpression of a dominant-negative mutant of ARF6 inhibited Salmonella-induced PMN transmigration, which was due to a reduction in apical release of the PMN chemoattractant PEEC (pathogen-elicited epithelial chemoattractant), without affecting bacterial internalization. Furthermore, ARF6 and its effector phospholipase D (PLD) were both required for bacteria-induced translocation of protein kinase C (PKC) to membranes. These results describe a novel signal transduction pathway, in which Salmonella initiates an ARF6- and PLD-dependent lipid signaling cascade that, in turn, directs activation of PKC, release of PEEC, and subsequent transepithelial PMN movement.

Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D.

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility.

Expression of constitutively active Rab5 uncouples maturation of the Salmonella-containing vacuole from intracellular replication.

The enteric bacterial pathogen Salmonella typhimurium enters and proliferates within both phagocytic and non-phagocytic host cells. Upon entry, the bacteria reside in membrane-bound vacuoles (SCVs) that mature with time, as evidenced by the sequential loss of early endosomal markers, followed by the selective recruitment of a number of lysosomal membrane glycoproteins (LAMPs). This remodelling process renders the SCVs non-fusogenic with lysosomes and is also thought to create a vacuolar environment permissive for replication. We demonstrate that disruption of the endocytic pathway by the expression of a constitutively active form of the small GTPase rab5 (rab5Q79L) significantly altered the biogenesis of the SCVs without inhibiting bacterial replication in HeLa cells. Expression of rab5Q79L caused the retention of early endosomal markers on SCVs and early acquisition of LAMP2, and led to an increase in the kinetics of intracellular replication. We also demonstrate that a significant fraction of LAMP2 in SCVs is derived from the cell surface via endocytosis rather than via the biosynthetic route. Further, in fibroblasts lacking a functional AP3 adaptor complex, in which all newly synthesized LAMP is delivered to the cell surface, recruitment of LAMP to the SCVs remained unaffected. These findings raise the possibility that all the SCV-associated LAMP could be derived by endocytosis from the cell surface.

Activation of the luteinizing hormone/choriogonadotropin hormone receptor promotes ADP ribosylation factor 6 activation in porcine ovarian follicular membranes.

Previously we demonstrated in a cell-free ovarian follicular plasma membrane model that agonist-dependent desensitization of the luteinizing hormone/choriogonadotropin receptor (LH/CG R) is GTP-dependent, mimicked by the addition of ADP-ribosylation factor (ARF) nucleotide binding site opener, which acts as a guanine nucleotide exchange factor for ARFs 1 and 6, and selectively inhibited by synthetic N-terminal ARF6 peptides. We therefore sought direct evidence that activation of the LH/CG R promotes activation of ARF1 and/or ARF6. Using a classic ARF activation assay, the cholera toxin-catalyzed ADP-ribosylation of G alpha(s), results show that LH/CG R activation stimulates an ARF protein by a brefeldin A-independent mechanism. Synthetic N-terminal inhibitory ARF6 but not ARF1 peptide blocks LH/CG R-stimulated ARF activity. LH/CG R activation also promotes the binding of a photoaffinity GTP analog to a protein that migrates on one- and two-dimensional polyacrylamide gel electrophoresis with ARF6. These results suggest that ARF6 is the predominant ARF activated by the LH/CG R. To activate ARF6, the LH/CG R does not appear to signal through the C-terminal regions of G alpha(i) or G alpha(q) or through the second or third intracellular loops or the N terminus of the cytoplasmic tail of the LH/CG R. Although exogenous recombinant ARNO promotes only a small increase in ARF6 activation in the presence of activated LH/CG R, hCG-stimulated ARF6 activation is reduced to basal levels by catalytically inactive ARF nucleotide binding-site opener. These results provide direct evidence that LH/CG R activation leads to the activation of membrane-delimited ARF6.

The GTPase Rac1 selectively regulates Salmonella invasion at the apical plasma membrane of polarized epithelial cells.

The bacterial pathogen Salmonella typhimurium colonizes its animal hosts by inducing its internalization into intestinal epithelial cells. This process requires reorganization of the actin cytoskeleton of the apical plasma membrane into elaborate membrane ruffles that engulf the bacteria. Members of the Rho family of small GTPases are critical regulators of actin structure, and in nonpolarized cells, the GTPase Cdc42 has been shown to modulate Salmonella entry. Because the actin architecture of epithelial cells is organized differently from that of nonpolarized cells, we examined the role of two Rho family GTPases, Cdc42 and Rac1, in invasion of polarized monolayers of MDCK cells by S. typhimurium. Surprisingly, we found that endogenous Rac1, but not Cdc42, was activated during bacterial entry at the apical pole, and that this activation required the bacterial effector protein SopE. Furthermore, expression of dominant inhibitory Rac1 but not Cdc42 significantly inhibited apical internalization of Salmonella, indicating that Rac1 activation is integral to the bacterial entry process. In contrast, during basolateral internalization, both Cdc42 and Rac1 were activated; however, neither GTPase was required for entry. These findings, which differ significantly from previous observations in nonpolarized cells, indicate that the host cell signaling pathways activated by bacterial pathogens may vary with cell type, and in epithelial tissues may further differ between plasma membrane domains.

Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes.

Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules. Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes. This mechanism may play an important role in the pH-dependent regulation of receptor-mediated endocytosis in proximal tubules in situ.

Desensitization of the luteinizing hormone/choriogonadotropin receptor in ovarian follicular membranes is inhibited by catalytically inactive ARNO(+).

We have investigated the participation of endogenous ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) in desensitization of the luteinizing hormone/choriogonadotropin (LH/CG) receptor, independent of receptor internalization, using a cell-free plasma membrane model. We recently showed that the addition of recombinant ARNO promotes binding of beta-arrestin1 to the third intracellular (3i) loop of the active LH/CG receptor, thereby reducing the ability of the receptor to activate the stimulatory G protein and signal to adenylyl cyclase. In the present report we determined whether ARNO is detectable in follicular membranes and whether the catalytically inactive E156K ARNO mutant, containing a mutation in the Sec7 domain, can act in a dominant negative manner to block LH/CG receptor desensitization. Results show that ARNO is readily detected in follicular membranes and that levels of membrane-associated ARNO increase with follicular maturation. The addition of catalytically inactive E156K ARNO blocks both the release of beta-arrestin1 from its membrane docking site, based on Western blot analysis, and development of LH/CG receptor desensitization. We also investigated whether a point mutation in the pleckstrin homology (PH) domain of ARNO (R280D), which blocks binding of phosphoinositides like phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate (PIP(2)) but not catalytic activity, disrupts LH/CG receptor desensitization. R280D ARNO neither promotes nor inhibits LH/CG receptor desensitization, consistent with a requirement of the PH domain of ARNO for its association with the plasma membrane. LH/CG receptor activation of ARNO is not mediated by activation of phosphatidylinositol 3-kinase (PI 3-kinase) or by G protein beta gamma subunits. Taken together, these results suggest that LH/CG receptor promotes beta-arrestin1 release from its membrane docking site to bind to the 3i loop of the LH/CG receptor via activation of membrane delimited endogenous ARNO. As ARNO activation is independent of PI 3-kinase and G beta gamma, our results are consistent with a role for PIP(2) in receptor-stimulated ARNO activation.

Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25.

Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.

The ADP ribosylation factor nucleotide exchange factor ARNO promotes beta-arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization.

Desensitization of guanine nucleotide binding protein-coupled receptors is a ubiquitous phenomenon characterized by declining effector activity upon persistent agonist stimulation. The luteinizing hormone/choriogonadotropin receptor (LH/CGR) in ovarian follicles exhibits desensitization of effector adenylyl cyclase activity in response to the mid-cycle surge of LH. We have previously shown that uncoupling of the agonist-activated LH/CGR from the stimulatory G protein (G(s)) is dependent on GTP and attributable to binding of beta-arrestin present in adenylyl cyclase-rich follicular membrane fraction to the third intracellular (3i) loop of the receptor. Here, we report that LH/CGR-dependent desensitization is mimicked by ADP ribosylation factor nucleotide-binding site opener, a guanine nucleotide exchange factor of the small G proteins ADP ribosylation factors (Arfs) 1 and 6, and blocked by synthetic N-terminal Arf6 peptide, suggesting that the GTP-dependent step of LH/CGR desensitization is receptor-dependent Arf6 activation. Arf activation by GTP and ADP ribosylation factor nucelotide-binding site opener promotes the release of docked beta-arrestin from the membrane, making beta-arrestin available for LH/CGR; Arf6 but not Arf1 peptides block beta-arrestin release from the membrane. Thus, LH/CGR appears to activate two membrane delimited signaling cascades via two types of G proteins: heterotrimeric G(s) and small G protein Arf6. Arf6 activation releases docked beta-arrestin necessary for receptor desensitization, providing a feedback mechanism for receptor self-regulation.

Identification of a plasma membrane-associated guanine nucleotide exchange factor for ARF6 in chromaffin cells. Possible role in the regulated exocytotic pathway.

ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6-GEF activity in the plasma membrane and in the cytosol of chromaffin cells. Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Using permeabilized chromaffin cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits, in a dose-dependent manner, both PLD activation and catecholamine secretion in calcium-stimulated cells. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K(+). The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.

Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors.

ARF proteins are important regulators of membrane dynamics and protein transport within the eukaryotic cell. The Sec7 domain is approximately 200 amino acids in size and stimulates guanine-nucleotide exchange on members of the ARF class of small GTPases. The members of one subclass of Sec7-domain proteins are direct targets of the secretion-inhibiting drug brefeldin A, which blocks the exchange reaction by trapping a reaction intermediate in an inactive, abortive complex. A separate subclass of Sec7-domain proteins is involved in signal transduction and possess a domain that mediates membrane binding in response to extracellular signals.

Recycling of AQP2 occurs through a temperature- and bafilomycin-sensitive trans-Golgi-associated compartment.

The exo- and endocytotic pathway in which aquaporin-2 (AQP2) travels between the plasma membrane and intracellular vesicles is only partially characterized. It is known that the antidiuretic hormone vasopressin induces a translocation of AQP2 from an intracellular to a plasma membrane location, both in kidney collecting duct principal cells and in transfected epithelial cells. Here we provide evidence suggesting that while AQP2 shifts from an intracellular location to the cell surface in response to vasopressin, AQP2 also constitutively recycles through a similar pathway in transfected LLC-PK(1) cells even in the absence of hormonal stimulation. Incubating cells at 20 degrees C blocks AQP2 recycling in a perinuclear compartment, regardless of whether vasopressin is present. The H(+)-ATPase inhibitor bafilomycin A1 also blocks the recycling pathway of AQP2 in a perinuclear compartment adjacent to the Golgi in the presence and absence of vasopressin stimulation, indicating a role of vesicle acidification in both the constitutive and regulated recycling of AQP2. Colocalization of AQP2 with clathrin, but not with giantin, after both bafilomycin treatment and a 20 degrees C block suggests that the compartment in which recycling AQP2 is blocked may be the trans-Golgi, and not cis- and medial-Golgi cisternae.

Regulation of ARNO nucleotide exchange by a PH domain electrostatic switch.

ARNO is a member of a family of guanine nucleotide exchange factors that activate small GTPases called ADP-ribosylation factors (ARFs) [1] [2] [3], which regulate vesicular trafficking and, in one case (ARF6), also regulate cortical actin structure [4]. ARNO is located at the plasma membrane, and in the presence of activated protein kinase C (PKC) can induce cortical actin rearrangements reminiscent of those produced by active ARF6 [5] [6] [7] [8]. High-affinity binding of ARNO to membranes, which is required for exchange activity, is mediated cooperatively by a pleckstrin homology (PH) domain and an adjacent carboxy-terminal polybasic domain [3] [9]. ARNO is phosphorylated in vivo by PKC on a single serine residue, S392, located within the carboxy-terminal polybasic domain. Mutation of S392 to alanine does not prevent ARNO-mediated actin rearrangements, suggesting that phosphorylation does not lead to ARNO activation [6]. Here, we report that phosphorylation negatively regulates ARNO exchange activity through a 'PH domain electrostatic switch'. Introduction of a negatively charged phosphate into the polybasic domain reduced interaction of ARNO with membranes both in vitro and in vivo, and inhibited exchange in vitro. This regulated membrane association is similar to the myristoyl electrostatic switch that controls membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) [10], but to our knowledge is the first demonstration of an electrostatic switch regulating the membrane interaction of a protein containing a PH domain. This mechanism allows regulation of ARNO lipid binding and exchange activity at two levels, phosphoinositide-dependent recruitment and PKC-dependent displacement from the membrane.

Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells.

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO.

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.

US family physicians' experiences with practice guidelines.

BACKGROUND: Practice guidelines were developed to improve medical outcomes and cost-effectiveness. The experiences of family physicians, who may need to use multiple guidelines in their practices, are crucial for effective development and implementation of practice guidelines. We surveyed a national sample of US family physicians about factors that affect their adoption and use of practice guidelines. METHODS: We mailed a structured survey to a national random sample of 400 family physician members of the American Academy of Family Physicians. RESULTS: The response rate was 51%. Most respondents (69%) reported a positive attitude about practice guidelines, but only 44% reported using any guidelines. More younger physicians thought that guidelines could be useful tools. Most preferred guidelines that could be modified (87%) and that were no longer than two pages. Only 27% of respondents knew where to locate a guideline on a particular topic. Forty-three percent of respondents reported that it would be useful if guidelines were a component of an electronic medical record. CONCLUSIONS: If guidelines are to be used by practicing family physicians, a generalist perspective needs to be considered in future guideline development and implementation. Younger physicians had more positive attitudes toward guidelines.

ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6.

ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of alphaLbeta2 integrins at the plasma membrane of lymphocytes.

Cloning and characterization of human phosphomannomutase, a mammalian homologue of yeast SEC53.

Phosphomannomutase (PMM) catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate, which is a substrate for the synthesis of GDP-mannose. This nucleotide sugar is then used in the synthesis of dolichol-phosphate-mannose, which is essential for N-linked glycosylation and thus the secretion of several glycoproteins as well as for the synthesis of glycosyl-phosphatidyl-inositol (GPI) anchored proteins. In the yeast Saccharomyces cerevisiae, SEC53, a gene required for viability, encodes PMM. Given the importance of PMM in glycoprotein synthesis, it is surprising that very little is known about the enzyme in higher eukaryotes. Recently, an autosomal recessive human disease, Carbohydrate-deficient glycoprotein syndrome type I (CDGS-I) has been correlated with severely reduced PMM activity. Here we report the isolation of a cDNA encoding human PMM, a protein of 29 kDa that is 55% identical and 66% similar to yeast Sec53p. Northern blot analysis shows a single 1.4 kb transcript that is ubiquitously expressed, although levels vary markedly among tissues. Expression of the human cDNA in a temperature-sensitive mutant sec53 yeast strain confers growth at the restrictive temperature, strongly suggesting that this gene encodes a functional PMM. Finally, when expressed in BHK cells, PMM is localized exclusively to the cytosol corresponding to its localization in yeast.