A quarter of patients with type 1 diabetes have co-existing non-islet autoimmunity: the findings of a UK population-based family study.

Individuals with type 1 diabetes (T1D) are at increased risk of coeliac disease (CD), autoimmune thyroiditis and autoimmune gastritis, but the absolute risks are unclear. The aim of this study was to investigate the prevalence of autoantibodies to tissue transglutaminase (TGA), thyroid peroxidase (TPOA) and gastric H+ /K+ -ATPase (ATPA) and their genetic associations in a well-characterized population-based cohort of individuals with T1D from the Bart's-Oxford family study for whom islet autoantibody prevalence data were already available. Autoantibodies in sera from 1072 patients (males/females 604/468; median age 11·8 years, median T1D duration 2·7 months) were measured by radioimmunoassays; HLA class II risk genotype was analysed in 973 (91%) using polymerase chain reaction with sequence specific primers (PCR-SSP). The prevalence of TGA (and/or history of CD), TPOA and ATPA in patients was 9·0, 9·6 and 8·2%, respectively; 3·1% had two or more autoantibodies. Females were at higher risk of multiple autoimmunity; TGA/CD were associated with younger age and TPOA with older age. ATPA were uncommon in patients under 5 years, and more common in older patients. Anti-glutamate decarboxylase autoantibodies were predictive of co-existing TPOA/ATPA. TGA/CD were associated with human leucocyte antigen (HLA) DR3-DQ2, with the DR3-DQ2/DR3-DQ2 genotype conferring the highest risk, followed by DR4-DQ8/DR4-DQ8. ATPA were associated with DR3-DQ2, DRB1*0404 (in males) and the DR3-DQ2/DR4-DQ8 genotype. TPOA were associated with the DR3-DQ2/DR3-DQ2 genotype. Almost one-quarter of patients diagnosed with T1D aged under 21 years have at least one other organ-specific autoantibody. HLA class II genetic profiling may be useful in identifying those at risk of multiple autoimmunity.

Zinc transporter (ZnT)8(186-194) is an immunodominant CD8+ T cell epitope in HLA-A2+ type 1 diabetic patients.

AIMS/HYPOTHESIS: Anti-zinc transporter (ZnT)8 autoantibodies are commonly detected in type 1 diabetic patients. We hypothesised that ZnT8 is also recognised by CD8(+) T cells and aimed to identify HLA-A2 (A*02:01)-restricted epitope targets. METHODS: Candidate epitopes were selected by ZnT8 plasmid DNA immunisation of HLA-A2/DQ8 transgenic mice and tested for T cell recognition in peripheral blood mononuclear cells of type 1 diabetic, type 2 diabetic and healthy participants by IFN-γ enzyme-linked immunospot. RESULTS: White HLA-A2(+) adults (83%) and children (60%) with type 1 diabetes displayed ZnT8-reactive CD8(+) T cells that recognised a single ZnT8(186-194) (VAANIVLTV) epitope. This ZnT8(186-194)-reactive fraction accounted for 50% to 53% of total ZnT8-specific CD8(+) T cells. Another sequence, ZnT8(153-161) (VVTGVLVYL), was recognised in 20% and 25% of type 1 diabetic adults and children, respectively. Both epitopes were type 1 diabetes-specific, being marginally recognised by type 2 diabetic and healthy participants (7-12% for ZnT8(186-194), 0% for ZnT8(153-161)). CONCLUSIONS/INTERPRETATION: ZnT8-reactive CD8(+) T cells are predominantly directed against the ZnT8(186-194) epitope and are detected in a majority of type 1 diabetic patients. The exceptional immunodominance of ZnT8(186-194) may point to common environmental triggers precipitating beta cell autoimmunity.

Development of a novel autoantibody assay for autoimmune gastritis in type 1 diabetic individuals.

BACKGROUND: Autoimmune atrophic body gastritis (ABG) and pernicious anaemia are prototypical, organ-specific autoimmune diseases whose prevalence in the general population is 2.0 vs 2 and 0.15-1%, respectively. The incidence of disease increases with age and is frequently associated with other autoimmune disorders such as type 1 diabetes mellitus (T1DM). Early diagnosis of ABG/pernicious anaemia is essential for the prevention and/or treatment before manifestations of chronic disease become irreversible. Parietal cell autoantibody detection via enzyme-linked immunosorbent assay is currently the most widely used biomarker of disease with diagnosis confirmed by subsequent immunohistochemistry via biopsy. METHODS: To improve the assay we designed a specific, molecularly defined radioimmunoprecipitation assay for early detection of ABG, targeting its major antigen, the gastric H+/K+ ATPase 4A subunit ATP4A. RESULTS: The major antigenic domain in ATP4A was tested against a panel of sera from new onset patients with T1DM which tested positive for the gold standard T1DM autoantibodies (IAA, IA2A, GAD65A, and ZnT8A). Significant immunoreactivity to ATP4A was measured (25%) while 6% of first-degree relatives of subjects with T1DM who were sero-negative for T1DM autoantigens were positive for ATP4A autoantibodies. ATP4A antibody prevalence increased with age of onset of T1DM, which is atypical of other T1DM autoantibodies. Immunoreactivity to ATP4A, unlike that of T1DM antigens, demonstrates a significant gender bias in newly diagnosed individuals with T1DM. CONCLUSION: Although the utility of the assay as a biomarker for T1DM is likely limited, it may serve as an improved indicator of ABG.

Kinetics of the post-onset decline in zinc transporter 8 autoantibodies in type 1 diabetic human subjects.

CONTEXT: Zinc transporter 8 (ZnT8) is a newly discovered islet autoantigen in human type 1A diabetes (T1D). OBJECTIVE: The objective was to document changes in ZnT8 autoantibody (ZnT8A) titer and prevalence after onset of disease in relationship to 65 kDa glutamate decarboxylase antibody (GADA) and islet cell antigen antibody (IA2A). DESIGN/PATIENTS: Autoantibody radioimmunoprecipitation assays were performed on sera from three groups: 21 individuals monitored every 3 months from diagnosis for 2.5 yr; 61 individuals monitored at six monthly intervals for 5-12 yr; and a cross-sectional study of 424 patients with T1D of 20-57 yr duration. Circulating C-peptide was determined as an index of residual ß-cell function. RESULTS: ZnT8A titers declined exponentially from clinical onset of T1D with a t(1/2) ranging from 26 to 530 wk, similar to C-peptide (23-300 wk). Life-table analysis of antibody prevalence to 12 yr indicated that ZnT8A measured with either Arg325 or Trp325 probes persisted for a shorter interval than IA2A. Although prevalence of ZnT8A, IA2A, and GADA were comparable at disease onset (70.4 vs. 73.4 vs. 64%), only 6.7% of individuals remained ZnT8A positive after 25 yr compared with 19.5% for IA2A and 25.9% for GADA. Titers of ZnT8A and IA2A in seropositive individuals decreased progressively, whereas GADA remained elevated consistent with periodic reactivation of GADA humoral autoimmunity. CONCLUSIONS: ZnT8 humoral autoreactivity declines rapidly in the first years after disease onset and is less persistent than IA2A or GADA in the longer term. ZnT8A determination may be a useful measure of therapeutic efficacy in the context of immune-based clinical interventions.

Cytokine-mediated induction of anti-apoptotic genes that are linked to nuclear factor kappa-B (NF-kappaB) signalling in human islets and in a mouse beta cell line.

AIMS/HYPOTHESIS: The destruction of pancreatic beta cells leading to type 1 diabetes in humans is thought to occur mainly through apoptosis and necrosis induced by activated macrophages and T cells, and in which secreted cytokines play a significant role. The transcription factor nuclear factor kappa-B (NF-kappaB) plays an important role in mediating the apoptotic action of cytokines in beta cells. We therefore sought to determine the changes in expression of genes modulated by NF-kappaB in human islets exposed to a combination of IL1beta, TNF-alpha and IFN-gamma. METHODS: Microarray and gene set enrichment analysis were performed to investigate the global response of gene expression and pathways modulated in cultured human islets exposed to cytokines. Validation of a panel of NF-kappaB-regulated genes was performed by quantitative RT-PCR. The mechanism of induction of BIRC3 by cytokines was examined by transient transfection of BIRC3 promoter constructs linked to a luciferase gene in MIN6 cells, a mouse beta cell line. RESULTS: Enrichment of several metabolic and signalling pathways was observed in cytokine-treated human islets. In addition to the upregulation of known pro-apoptotic genes, a number of anti-apoptotic genes including BIRC3, BCL2A1, TNFAIP3, CFLAR and TRAF1 were induced by cytokines through NF-kappaB. Significant synergy between the cytokines was observed in NF-kappaB-mediated induction of the promoter of BIRC3 in MIN6 cells. CONCLUSIONS/INTERPRETATION: These findings suggest that, via NF-kappaB activation, cytokines induce a concurrent anti-apoptotic pathway that may be critical for preserving islet integrity and viability during the progression of insulitis in type 1 diabetes.

Association between anti-ZnT8 autoantibody specificities and SLC30A8 Arg325Trp variant in Japanese patients with type 1 diabetes.

AIMS/HYPOTHESIS: We analysed the association between humoral autoreactivity to zinc transporter-8 (ZnT8) and the SLC30A8 rs13266634 polymorphism (Arg325Trp), which is located at the most distal loop in the ZnT8 protein. METHODS: Autoantibodies to ZnT8 were determined by RIA in 270 patients with type 1 diabetes using ZnT8 carboxy-terminal constructs (amino acids 268-369) carrying 325Trp(CW) and 325Arg(CR) and a hybrid construct (CW-CR). Forty-four ZnT8 autoantibody-positive sera with genomic DNA were used to examine the association between reactivity to ZnT8 constructs and the rs13266634 genotype. RESULTS: Seventy-five patients reacted to the CW-CR hybrid construct, whereas 37 and 36 patients reacted to the CW and CR constructs, respectively. All sera positive for either CW or CR autoantibodies were positive for CW-CR autoantibodies. Among 19 patients with a 325Arg(CC) genotype, 5% had CW-specific autoantibodies, 42% had CR-specific autoantibodies and 32% had dual reactivity. Conversely, 73% of 15 patients with the 325Trp(TT) genotype had CW-specific autoantibodies, no patients had CR-specific autoantibodies and 13% had dual reactivity. Nine of the ten patients (90%) with the CT genotype reacted with either CR or CW constructs. The titre of CR autoantibodies in patients carrying the C allele was significantly higher than that in TT homozygotes (p < 0.0001). In contrast, the titre of CW autoantibodies in patients carrying a T allele was significantly higher than that in CC homozygotes (p < 0.005). No evidence of an association between rs13266634 and type 1 diabetes was observed. CONCLUSIONS/INTERPRETATION: These results indicate that variant residue at amino acid 325 is a key determinant of humoral autoreactivity to ZnT8 and that the SLC30A8 genotype is an important determinant of autoantibody specificity.

Conserved epitopes in the protein tyrosine phosphatase family of diabetes autoantigens.

IA2 and phogrin are important targets of humoral and cell-mediated autoimmunity in type 1 diabetes in man. They belong to a conserved subfamily of transmembrane protein tyrosine phosphatases (PTPs) associated with the regulatory pathway of secretion. To examine potential cross-reactivity between PTP family members we tested sera from T1D patients for reactivity to IA2, and the Drosophila (FLYDA) and C. elegans (IDA) orthologs using radioimmunoprecipitation assays of (35)S Met-labeled in vitro translated products of the cytosolic domains of these proteins. Approximately 80% of sera reacted with at least one probe. Of these, 82.5% showed reactivity to human IA2, 74.1% to FLYDA, and 33.7% to IDA. The majority of sera that bound FLYDA and/or IDA also recognized IA2. This raises the possibility that in some cases reactivity to IA2 may have arisen by molecular mimicry.

Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation.

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3'-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

In vitro assays of vesicular transport.

Movement of proteins and lipids between the various compartments of eukaryotic cells is fundamental to the maintenance of cellular homeostasis, and an understanding of the molecular mechanisms that govern these processes remains a key goal of cell biological research. This aim has been greatly facilitated by the development of assays that recapitulate specific events in vitro. In the following article we provide an overview of some of the currently used assays that measure the movement of proteins within the exocytic and endocytic pathways, and provide a starting point for those wishing to establish their own systems to study other vesicular transport steps.

Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation.

Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxypeptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin, which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mammalian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug.

Loss of cation-independent mannose 6-phosphate receptor expression promotes the accumulation of lysobisphosphatidic acid in multilamellar bodies.

A number of recent studies have highlighted the importance of lipid domains within endocytic organelles in the sorting and movement of integral membrane proteins. In particular, considerable attention has become focussed upon the role of the unusual phospholipid lysobisphosphatidic acid (LBPA). This lipid appears to be directly involved in the trafficking of cholesterol and glycosphingolipids, and accumulates in a number of lysosomal storage disorders. Antibody-mediated disruption of LBPA function also leads to mis-sorting of cation-independent mannose 6-phosphate receptors. We now report that the converse is also true, and that spontaneous loss of cation-independent mannose 6-phosphate receptors from a rat fibroblast cell line led to the formation of aberrant late endocytic structures enriched in LBPA. Accumulation of LBPA was directly dependent upon the loss of the receptors, and could be reversed by expression of bovine cation-independent mannose 6-phosphate receptors in the mutant cell line. Ultrastructural analysis indicated that the abnormal organelles were electron-dense, had a multi-lamellar structure, accumulated endocytosed probes, and were distinct from dense-core lysosomes present within the same cells. The late endocytic structures present at steady state within any particular cell likely reflect the balance of membrane traffic through the endocytic pathway of that cell, and the rate of maturation of individual endocytic organelles. Moreover, there is considerable evidence which suggests that cargo receptors also play a direct mechanistic role in membrane trafficking events. Therefore, loss of such a protein may disturb the overall equilibrium of the pathway, and hence cause the accumulation of aberrant organelles. We propose that this mechanism underlies the phenotype of the mutant cell line, and that the formation of inclusion bodies in many lysosomal storage diseases is also due to an imbalance in membrane trafficking within the endocytic pathway.

Direct transport of newly synthesized HLA-DR from the trans-Golgi network to major histocompatibility complex class II containing compartments (MIICS) demonstrated using a novel tyrosine-sulfated chimera.

Binding of antigenic peptides to major histocompatibility complex (MHC) class II glycoproteins occurs in specialized endocytic compartments of antigen-presenting cells, which in man are termed MIICs. Newly synthesized MHC class II molecules are transported from the trans-Golgi network to MIICs, but previous studies of this important step in antigen processing have failed to conclusively determine whether most immature MHC class II complexes are transported directly to the processing compartments or are first transiently exposed at the cell surface. To attempt to resolve this question, I constructed a chimeric HLA-DRalpha chain containing two optimal tyrosine sulfation motifs. When expressed in a human B lymphoblastoid cell line lacking functional DRalpha chains, the chimera was correctly incorporated into complexes containing endogenous beta and invariant chains, transported to the trans-Golgi network, and efficiently sulfated. Pulse-chase experiments showed that the sulfated complexes were rapidly transported to processing compartments with kinetics consistent with direct transport from the trans-Golgi network. The rate of maturation was not significantly altered in cells expressing a temperature-sensitive mutant of dynamin under conditions where the endocytosis of transferrin was inhibited by 95%, confirming that endocytosis was not required for delivery to MIICs. Maturation of MHC class II-containing complexes was inhibited by aluminum fluoride and brefeldin A, indicating the involvement of heterotrimeric G-proteins and ADP-ribosylation factor in the transport event(s). The procedure described provides a unique mechanism to study critical events in antigen processing and presentation.

Phosphoinositide 3-kinases and membrane traffic.

Phosphoinositide 3-kinases (PI 3-kinases) and their 3-phosphoinositide products were identified initially as components of intracellular signalling pathways emanating from cell surface receptors. A new role for 3-phosphoinositides in the constitutive movement o f proteins from one intracellular compartment to another was proposed with the discovery of homology between the product of a yeast gene important for vacuolar sorting, Vps34p, and a mammalian PI 3-kinase. Recent studies have implicated PI 3-kinase as an essential component in membrane traffic at specific steps o f the trans-Golgi-network-endosomal pre-lysosomal system. Evidence largely emerging from the insulin-stimulated glucose transport system suggests that PI 3-kinase may also mediate the effects o f growth factors on membrane traffic events. These studies suggest a possible link between growth-factor-stimulated and constitutive membrane traffic in the endosomal system.

Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes.

At present little is known of the biochemical machinery controlling transport of newly synthesized lysosomal hydrolases from the trans-Golgi network (TGN) to endosomes. The demonstration that Vps34p (a protein required for targeting soluble hydrolases to the vacuole in Saccharomyces cerevisiae) is a phosphatidylinositol 3-kinase (PI3-K) suggested the possibility that a homologous enzyme might be involved in the equivalent step in mammalian cells. Using the PI3-K inhibitors wortmannin and LY294002, I provide evidence to support this hypothesis. Treatment of K-562 cells with wortmannin induced secretion of procathepsin D, with half-maximal inhibition of accurate targeting to lysosomes at 10-20 nM. Kinetic analysis indicated that a late Golgi (TGN) step was affected, and that other constitutive vesicular transport events were not. The M6P recognition signal was still generated in the presence of wortmannin suggesting that the drug was directly inhibiting export of the receptor-ligand complex from the TGN, while removal of the drug led to a rapid restoration of accurate sorting. At the concentrations used, wortmannin and LY294002 are presently accepted to be specific inhibitors of PI3-K. I conclude that these data implicate such an enzyme in the trafficking of M6P-receptor-ligand complexes from the TGN towards lysosomes.

A GDP-bound of rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments.

Rab1 is a small GTPase regulating vesicular traffic between early compartments of the secretory pathway. To explore the role of rab1 we have analyzed the function of a mutant (rab1a[S25N]) containing a substitution which perturbs Mg2+ coordination and reduces the affinity for GTP, resulting in a form which is likely to be restricted to the GDP-bound state. The rab1a(S25N) mutant led to a marked reduction in protein export from the ER in vivo and in vitro, indicating that a guanine nucleotide exchange protein (GEP) is critical for the recruitment of rab1 during vesicle budding. The mutant protein required posttranslational isoprenylation for inhibition and behaved as a competitive inhibitor of wild-type rab1 function. Both rab1a and rab1b (92% identity) were able to antagonize the inhibitory activity of the rab1a(S25N) mutant, suggesting that these two isoforms are functionally interchangeable. The rab1 mutant also inhibited transport between Golgi compartments and resulted in an apparent loss of the Golgi apparatus, suggesting that Golgi integrity is coupled to rab1 function in vesicular traffic.

Rab1 and Ca2+ are required for the fusion of carrier vesicles mediating endoplasmic reticulum to Golgi transport.

Members of the rab/YPT1/SEC4 gene family of small molecular weight GTPases play key roles in the regulation of vesicular traffic between compartments of the exocytic pathway. Using immunoelectron microscopy, we demonstrate that a dominant negative rab1a mutant, rab1a(N124I), defective for guanine nucleotide binding in vitro, leads to the accumulation of vesicular stomatitis virus glycoprotein (VSV-G) in numerous pre-cis-Golgi vesicles and vesicular-tubular clusters containing rab1 and beta-COP, a subunit of the coatomer complex. Similar to previous observations (Balch et al. 1994. Cell. 76:841-852), VSV-G was concentrated nearly 5-10-fold in vesicular carriers that accumulate in the presence of the rab1a(N124I) mutant. VSV-G containing vesicles and vesicular-tubular clusters were also found to accumulate in the presence of a rab1a effector domain peptide mimetic that inhibits endoplasmic reticulum to Golgi transport, as well as in the absence of Ca2+. These results suggest that the combined action of a Ca(2+)-dependent protein and conformational changes associated with the GTPase cycle of rab1 are essential for a late targeting/fusion step controlling the delivery of vesicles to Golgi compartments.

Differential inhibition of multiple vesicular transport steps between the endoplasmic reticulum and trans Golgi network.

Using the glycoprotein of the tsO45 mutant of vesicular stomatitis virus (VSV-G) as a marker, we have developed a system capable of measuring vesicular transport from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) in vitro. Movement from the ER to the cis Golgi compartment was assessed by the conversion of VSV-G from a totally endoglycosidase D (endo D)-resistant form to a species containing one endo D-resistant and one endo D-sensitive oligosaccharide (GD1). Similarly, delivery to the medial cisternae was measured by the appearance of the completely endo D-sensitive form of VSV-G (GD2) or by the acquisition of complete resistance to endoglycosidase H (endo H) (GHr) and delivery to the TGN by the appearance of an endo H-resistant form of VSV-G which was sensitive to digestion with neuraminidase and subsequently beta-galactosidase (GHt). Movement between each sequential compartment required ATP and soluble proteins (cytosol) and was inhibited by nonhydrolyzable analogues of GTP and by an antibody toward the N-ethylmaleimide-sensitive factor NSF. In contrast, fractionation of the cytosol by ammonium sulfate precipitation indicated that distinct proteins were required for movement between successive compartments. Similarly, inclusion of a mutant form of the small molecular weight GTP-binding protein rab1A inhibited movement between the ER and cis Golgi, and between the cis and medial cisternae, but did not affect transport from the medial Golgi to the TGN. Conversely, the protein kinase inhibitor staurosporine prevented movement between the medial Golgi and the TGN but did not influence transport between the ER and early Golgi compartments. This study provides the first demonstration that vesicular transport between the ER and TGN can be reconstituted in a cytosol-dependent fashion in vitro, allowing a direct analysis of the roles of individual components in multiple transport events.

Morphological analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: role of the P58 containing compartment.

The glycoside digitonin was used to selectively permeabilize the plasma membrane exposing functionally and morphologically intact ER and Golgi compartments. Permeabilized cells efficiently transported vesicular stomatitis virus glycoprotein (VSV-G) through sealed, membrane-bound compartments in an ATP and cytosol dependent fashion. Transport was vectorial. VSV-G protein was first transported to punctate structures which colocalized with p58 (a putative marker for peripheral punctate pre-Golgi intermediates and the cis-Golgi network) before delivery to the medial Golgi compartments containing alpha-1,2-mannosidase II and processing of VSV-G to endoglycosidase H resistant forms. Exit from the ER was inhibited by an antibody recognizing the carboxyl-terminus of VSV-G. In contrast, VSV-G protein colocalized with p58 in the absence of Ca2+ or the presence of an antibody which inhibits the transport component NSF (SEC18). These studies demonstrate that digitonin permeabilized cells can be used to efficiently reconstitute the early secretory pathway in vitro, allowing a direct comparison of the morphological and biochemical events involved in vesicular tafficking, and identifying a key role for the p58 containing compartment in ER to Golgi transport.

Evidence for the regulation of exocytic transport by protein phosphorylation.

We investigated the effects of the protein phosphatase inhibitors okadaic acid and microcystin-LR upon transport of newly synthesized proteins through the exocytic pathway. Treatment of CHO cells with 1 microM okadaic acid rapidly inhibited movement of a marker protein (vesicular stomatitis virus G protein) from the endoplasmic reticulum to the Golgi compartment. Both okadaic acid and microcystin-LR also inhibited transport in an in vitro assay reconstituting movement to the Golgi compartment, at concentrations equivalent to those required to inhibit phosphorylase phosphatase activity. Inhibition both in vivo and in vitro could be antagonized by protein kinase inhibitors, suggesting that protein phosphorylation was directly responsible for this effect. An early stage in the transport reaction associated with vesicle formation or targeting was inhibited by protein phosphorylation, which could be reversed by fractions enriched in protein phosphatase 2A. Protein kinase antagonists did not inhibit transport between sequential compartments of the exocytic pathway in vitro, suggesting that protein phosphorylation is not itself required for vesicular transport. During mitosis, vesicular transport is inhibited simultaneous to the activation of maturation-promoting factor. It is proposed that the inhibition caused by okadaic acid and microcystin-LR involves a similar mechanism to that responsible for the mitotic arrest of vesicular transport.