Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi

Reticulons (RTNs) constitute a family of endoplasmic reticulum (ER)-associated proteins with a reticular distribution. Despite the implication of their neuronal isoforms in axonal regeneration, the function of their widely expressed isoforms is largely unknown. In this study, we examined the role of the ubiquitously expressed RTN3 in membrane trafficking. Ectopically expressed RTN3 exhibited heterogeneous patterns; filamentous, reticular, and granular distributions. The ER morphology changed accordingly. In cells where RTN3 displayed a filamentous/reticular distribution, protein transport between the ER and Golgi was blocked, and Golgi proteins were dispersed. In contrast, ERGIC-53, a marker for the ER-Golgi intermediate compartment, accumulated at the perinuclear region, and remained there even after cells were treated with agents that induce redistribution of Golgi proteins to the ER, indicating an inhibition of Golgi-to-ER transport of ERGIC-53. These results suggest that RTN3 plays a role in membrane trafficking in the early secretory pathway.     

Implication of ZW10 in membrane trafficking between the endoplasmic reticulum and Golgi

ZW10, a dynamitin-interacting protein associated with kinetochores, is known to participate directly in turning off of the spindle checkpoint. In the present study, we show that ZW10 is located in the endoplasmic reticulum as well as in the cytosol during interphase, and forms a subcomplex with RINT-1 (Rad50-interacting protein) and p31 in a large complex comprising syntaxin 18, an endoplasmic reticulum-localized t-SNARE implicated in membrane trafficking. Like conventional syntaxin-binding proteins, ZW10, RINT-1 and p31 dissociated from syntaxin 18 upon Mg(2+)-ATP treatment in the presence of NSF and alpha-SNAP, whereas the subcomplex was not disassembled. Overexpression, microinjection and knockdown experiments revealed that ZW10 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. The present results disclose an unexpected role for a spindle checkpoint protein, ZW10, during interphase.     

MAP1B‐LC1 prevents autophagosome formation by linking syntaxin 17 to microtubules

In fed cells, syntaxin 17 (Stx17) is associated with microtubules at the endoplasmic reticulum–mitochondria interface and promotes mitochondrial fission by determining the localization and function of the mitochondrial fission factor Drp1. Upon starvation, Stx17 dissociates from microtubules and Drp1, and binds to Atg14L, a subunit of the phosphatidylinositol 3‐kinase complex, to facilitate phosphatidylinositol 3‐phosphate production and thereby autophagosome formation, but the mechanism underlying this phenomenon remains unknown. Here we identify MAP1B‐LC1 (microtubule‐associated protein 1B‐light chain 1) as a critical regulator of Stx17 function. Depletion of MAP1B‐LC1 causes Stx17‐dependent autophagosome accumulation even under nutrient‐rich conditions, whereas its overexpression blocks starvation‐induced autophagosome formation. MAP1B‐LC1 links microtubules and Stx17 in fed cells, and starvation causes the dephosphorylation of MAP1B‐LC1 at Thr217, allowing Stx17 to dissociate from MAP1B‐LC1 and bind to Atg14L. Our results reveal the mechanism by which Stx17 changes its binding partners in response to nutrient status.     

Association of the fusion protein NSF with clathrin-coated vesicle membranes.

N-ethylmaleimide-sensitive fusion protein (NSF) is a component of intracellular transport reactions. In order to understand the role of NSF during the fusion of endocytic transport vesicles with the endosome, we have investigated the binding of NSF to purified clathrin-coated vesicle components. First, we have examined whether detergent-solubilized coated vesicle membranes will support formation of NSF-containing 'fusion complexes'. Our results show that these membranes are substantially enriched in components capable of driving formation of these complexes, when compared with membranes from other sources. Secondly, we have analysed coated vesicle preparations for their NSF content. Coated vesicle preparations contain significant amounts of NSF. This was shown to be associated with coated vesicles rather than contaminating membranes by a number of criteria, and was found to be bound in an ATP-independent manner. These findings are discussed in the light of current models for vesicle fusion.     

Impaired expression of high affinity interleukin 2 receptor on activated lymphocytes from patients with systemic lupus erythematosus

Peripheral lymphocytes stimulated with phytohemagglutinin (PHA-blasts) were examined for their responsiveness to exogenous interleukin 2 (IL-2). The proliferative response of PHA-blasts to IL-2 was significantly lower in patients with systemic lupus erythematosus (SLE) than in normal subjects. To clarify the reason for this defect, the expression of IL-2 receptor (IL-2R) on PHA-blasts was investigated using anti-Tac antibody and purified IL-2. Cytofluorometric analysis showed no statistical differences in the Tac positivity of PHA-blasts among normal subjects and patients with active and inactive SLE. Scatchard analysis using 125I-labeled anti-Tac monoclonal antibody revealed that the number of Tac epitopes on PHA-blasts was also not different among them. Next, the affinity of IL-2R expressed on PHA-blasts was determined by Scatchard analysis using radiolabeled IL-2 as a ligand. The number of high affinity IL-2R on the PHA-blasts was significantly decreased in patients with active and inactive SLE, as compared with normal subjects. The responsiveness of PHA-blasts to exogenous IL-2 was well correlated to the number of high affinity IL-2R, but not to the number of Tac epitopes or total IL-2R. Inasmuch as high affinity components of IL-2R are functionally active, the defective expression of high affinity IL-2R may be responsible for the T cell dysfunctions in SLE.     

Affinity labeling of adenylate kinase with adenosine diphosphopyridoxal. Presence of Lys21 in the ATP-binding site

Adenosine diphosphopyridoxal, the affinity labeling reagent specific for a lysyl residue in the nucleotide-binding site of several enzymes (Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964; Tamura, J. K., Rakov, R. D., and Cross R. L. (1986) J. Biol. Chem. 261, 4126-4133) was applied to adenylate kinase from rabbit muscle. Incubation of the enzyme with a low concentration of the reagent at 25 degrees C for 20 min followed by reduction by sodium borohydride resulted in rapid inactivation of the enzyme. Extrapolation to 100% loss of enzyme activity gave a value of 1.0 mol of the reagent per mol of enzyme. ADP, ATP, and MgATP almost completely protected the enzyme from inactivation, whereas AMP offered little retardation of the inactivation. Dilution of the inactivated enzyme which had not been treated with the reducing reagent led to restoration of enzyme activity. This reactivation was accelerated by ATP but not by AMP. Structural study of the labeled peptide showed that Lys21 is exclusively labeled by adenosine diphosphopyridoxal. These results suggest that the epsilon-amino group of Lys21 is located in the ATP-binding site of the enzyme, more specifically at or close to the subsite for the gamma-phosphate of the nucleotide.     

Affinity labeling of the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by adenosine triphosphopyridoxal: identification of the reactive lysyl residue

Adenosine triphosphopyridoxal (AP3PL) was used as an affinity label directed toward the ATP binding site of the Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum (SR). The reagent inhibited the ATPase activity competitively with ATP, Ki = 20 microM. Incubation of SR membranes with 100 microM AP3PL followed by treatment with NaBH4 resulted in 90% inactivation of the E-P forming activity as well as of the Ca2+-transporting activity. Adenosine di- and tetraphosphopyridoxals had similar but less pronounced effects on the Ca2+-transport system. AP3PL was bound to ATPase in a one-to-one stoichiometry in parallel with the loss of the enzymatic activities. ATP and ADP prevented the binding of AP3PL and thereby protected the enzyme from inactivation. The SR membranes were labeled with [3H]AP3PL and then digested with thermolysin in order to identify the attachment site of the affinity label. A 3H-labeled peptide (Val-Glu-Pro-Ser-His-Lys* 684-Ser-Lys) was purified to homogeneity by Sephadex LH-20 chromatography and C18-reversed phase HPLC (Lys* denotes the binding site of [3H]AP3PL). These results indicate that the SR-ATPase peptide is folded in such a manner that Lys684 and Asp351, the phosphorylation site, are located very close to each other, since the distance between the 4-formyl group reacting with Lys684 and the gamma-phosphoryl group of the ATP moiety of AP3PL is rather small.     

Alpha-glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein

By using a synthetic oligonucleotide probe identical to a part of the gene for the Escherichia coli major outer membrane lipoprotein, we have cloned a gene from E. coli chromosomal DNA. However, the cloned gene was not one of the lipoprotein genes. The amino acid sequence deduced from its nucleotide sequence shows extensive similarities instead to alpha-glucan phosphorylase (EC 2.4.1.1). The gene, glgP, is located immediately downstream from glgA, the gene for glycogen synthase. The glgP gene was inserted into pUC9 vector and expressed in the presence of the lac inducer. The gene product was purified to apparent homogeneity as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In all chromatographies, the protein was eluted accompanied by a low phosphorylase activity. The final preparation showed phosphorolytic activity to various alpha-glucans, although the specific activity was extremely low compared to other alpha-glucan phosphorylases under the standard assay conditions. Its enzymatic activity, however, increased almost linearly as the concentration of glucan increased, reaching a value comparable with those of other phosphorylases. The amino acid sequence deduced was compared with those of alpha-glucan phosphorylases from other sources.     

Identification of the Neuroblastoma-amplified Gene Product as a Component of the Syntaxin 18 Complex Implicated in Golgi-to-Endoplasmic Reticulum Retrograde Transport

Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.