Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex

A bovine liver protein which catalyzes the transfer of triglyceride between membranes has previously been isolated from the lumen of the microsomal fraction. When further purified about 100-fold, two polypeptides of molecular mass 58,000 and 88,000 were identified (Wetterau, J. R., and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222). We demonstrate here that the two polypeptides (referred to as 58-kDa and 88-kDa, respectively) are associated in a protein-protein complex, and that the triglyceride transfer activity is associated with this complex. Antibodies specific for either polypeptide immunoprecipitated both the 58-kDa and 88-kDa polypeptides as well as the lipid transfer activity. The 58-kDa subunit of the microsomal transfer protein complex was identified as protein disulfide-isomerase (PDI) (EC 5.3.4.1) by 1) a comparison of the amino-terminal sequence of PDI and the 58-kDa subunit of the transfer protein, 2) a comparison of the reverse phase high performance liquid chromatography peptide maps of CNBr digests of PDI and the lipid transfer protein, 3) immunoprecipitation competition experiments in which PDI was found to compete with the lipid transfer protein for immunoprecipitation by the anti-58-kDa polyclonal antibodies, 4) immunological cross-reactivity of the microsomal triglyceride transfer protein complex with polyclonal antibodies raised against PDI, and 5) the appearance of protein disulfide isomerase activity following the dissociation of purified microsomal transfer protein complex with guanidine HCl. In conclusion, the microsomal triglyceride transfer protein has a multi-subunit structure which is unique compared to other intracellular lipid transfer proteins which have been described to be single polypeptides. The unexpected finding that PDI is a component of the microsomal triglyceride transfer protein complex suggests a new previously undescribed role for protein disulfide isomerase.     

The STT3 protein is a component of the yeast oligosaccharyltransferase complex

N-linked protein glycosylation is an essential process in eukaryotic cells. In the central reaction, the oligosaccharyltransferase (OTase) catalyzes the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ from dolicholpyrophosphate onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum. The product of the essential gene STT3 is required for OTase activity in vivo, but is not present in highly purified OTase preparations. Using affinity purification of a tagged Stt3 protein, we now demonstrate that other components of the OTase complex, namely Ost1p, Wbp1p and Swp1p, specifically co-purify with the Stt3 protein. In addition, different conditional stt3 alleles can be suppressed by overexpression of either OST3 and OST4, which encode small components of the OTase complex. These genetic and biochemical data show that the highly conserved Stt3p is a component of the oligosaccharyltransferase complex. Received: 3 June 1997 / Accepted: 29 July 1997     

Vaccinia virus G9 protein is an essential component of the poxvirus entry-fusion complex

The vaccinia virus G9R gene (VACWR087) encodes a protein of 340 amino acids with the following structural features that are conserved in all poxviruses: a site for N-terminal myristoylation, 14 cysteines, and a C-terminal transmembrane domain. Previous studies showed that G9 is one of eight proteins associated in a putative entry-fusion complex. Our attempt to isolate a mutant without the G9R gene was unsuccessful, suggesting that it is essential for virus replication. To further investigate its role, we constructed a recombinant vaccinia virus in which G9R is regulated by addition of an inducer. Induced G9 protein was associated with mature infectious virions and could be labeled with a membrane-impermeant biotinylation reagent, indicating surface exposure. Omission of inducer reduced the infectious-virus yield by about 1.5 logs; nevertheless, all stages of virus morphogenesis appeared normal and extracellular virions were present on the cell surface. Purified virions assembled without inducer had a specific infectivity of less than 5% of the normal level and a comparably small amount of G9, whereas their overall polypeptide composition, including other components of the entry-fusion complex, was similar to that of virions made in the presence of inducer or of wild-type virions. G9-deficient virions bound to cells, but penetration of cores into the cytoplasm and early viral RNA synthesis were barely detected, and cell-cell fusion was not triggered by low pH. Of the identified components of the multiprotein complex, G9 is the sixth that has been shown to be required for entry and membrane fusion.     

Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin

The dynactin complex visualized by deepetch electron microscopy appears as a short filament 37-nm in length, which resembles F-actin, plus a thinner, laterally oriented filament that terminates in two globular heads. The locations of several of the constituent polypeptides were identified on this structure by applying antibodies to decorate the dynactin complex before processing for electron microscopy. Antibodies to the actin-related protein Arp1 (previously referred to as actin- RPV), bound at various sites along the filament, demonstrating that this protein assembles in a polymer similar to conventional actin. Antibodies to the barbed-end actin-binding protein, capping protein, bound to one end of the filament. Thus, an actin-binding protein that binds conventional actin may also bind to Arp1 to regulate its polymerization. Antibodies to the 62-kD component of the dynactin complex also bound to one end of the filament. An antibody that binds the COOH-terminal region of the 160/150-kD dynactin polypeptides bound to the globular domains at the end of the thin lateral filament, suggesting that the dynactin polypeptide comprises at least part of the sidearm structure.     

N-acetylglucosamine transferase is an integral component of a kinesin-directed mitochondrial trafficking complex

Trafficking kinesin proteins (TRAKs) 1 and 2 are kinesin-associated proteins proposed to function in excitable tissues as adaptors in anterograde trafficking of cargoes including mitochondria. They are known to associate with N-acetylglucosamine transferase and the mitochondrial rho GTPase, Miro. We used confocal imaging, Förster resonance energy transfer and immunoprecipitations to investigate association between TRAKs1/2, N-acetylglucosamine transferase, the prototypic kinesin-1, KIF5C, and Miro. We demonstrate that in COS-7 cells, N-acetylglucosamine transferase, KIF5C and TRAKs1/2 co-distribute. Förster resonance energy transfer was observed between N-acetylglucosamine transferase and TRAKs1/2. Despite co-distributing with KIF5C and immunoprecipitations demonstrating a TRAK1/2, N-acetylglucosamine transferase and KIF5C ternary complex, no Förster resonance energy transfer was detected between N-acetylglucosamine transferase and KIF5C. KIF5C, N-acetylglucosamine transferase, TRAKs1/2 and Miro formed a quaternary complex. The presence of N-acteylglucosamine transferase partially prevented redistribution of mitochondria induced by trafficking proteins 1/2 and KIF5C. TRAK2 was a substrate for N-acetylglucosamine transferase with TRAK2 (S562) identified as a site of O-N-acetylglucosamine modification. These findings substantiate trafficking kinesin proteins as scaffolds for the formation of a multi-component complex involved in anterograde trafficking of mitochondria. They further suggest that O-glycosylation may regulate complex formation.     

The Leishmania donovani lipophosphoglycan T lymphocyte-reactive component is a tightly associated protein complex

Lymphocytes from mice immunized with Leishmania donovani (LPG) were specifically stimulated to proliferate in vitro by purified LPG or its delipidated congener, phosphoglycan. The response was dose dependent and required prior immunization with either LPG or phosphoglycan. Proliferation was eliminated by specific depletion of Thy-1+ cells with antisera and C and the proliferating T cell subset was shown to be CD4+CD8-. Tests of various LPG fragments indicated that the T cell stimulation was associated with the core structure of LPG rather than the lipid or phosphoglycan repeat structure. However, amino acid analysis of LPG and active LPG fragments, after acid hydrolysis, showed the presence of amino acids in peptide linkage. Specific hydrolysis of the glycosidic linkages in LPG with trifluoromethanesulfonic acid provided polypeptide material reactive with two mAb previously believed to be LPG carbohydrate core specific. The protein was separated from LPG by reverse phase chromatography and shown to be a complex of proteins with common epitopes recognized by the two mAb. The dominant species isolated from LPG was a set of small, approximately 11,000 Mr, molecules. Subsequent T cell proliferation studies showed that the lymphocyte stimulation was associated with the protein component of LPG and not the glycan.     

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.     

A complex from cultured Chinese hamster ovary cells containing nine aminoacyl-tRNA synthetases. Thermolabile leucyl-tRNA synthetase from the tsH1 mutant cell line is an integral component of this complex

The size distribution of the 20 aminoacyl-tRNA synthetases from wild-type Chinese hamster ovary (CHO) cells and from the mutant cell line tsH1, containing a temperature-sensitive leucyl-tRNA synthetase, was determined by gel filtration. Nine aminoacyl-tRNA synthetases, specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine and proline, which coeluted as high-Mr entities (Mr approximately 1.2 X 10(6)), were further co-purified to yield a multienzyme complex, the polypeptide composition of which was identical to that previously determined for the complex from rabbit liver. Immunoprecipitates obtained from crude extracts of wild-type and tsH1 mutant cells, using specific antibodies directed to the lysyl-tRNA or methionyl-tRNA synthetase components of the complex, displayed the same polypeptide compositions as that of the purified complex, thereby establishing the heterotypic nature of this complex. Although the activity of leucyl-tRNA synthetase from the mutant cells, grown at a permissive temperature, was low compared to that from the wild-type, the polypeptide of Mr 129 000, corresponding to this enzyme, was present in similar amounts and occurred exclusively as a component of the high-Mr complex. Finally, we report that attempts to demonstrate phosphorylation of the components of the complex from cultured CHO, HeLa and C3 cells were unsuccessful.     

Lactic acid is absorbed from the small intestine of sheep

A series of experiments was conducted in vivo on anaesthetized sheep to explore the hypothesis that lactic acid is absorbed from the small intestine of sheep. Test solutions varying in lactic acid concentration, pH, osmolarity, and with fixed physiological concentrations of volatile fatty acids (VFAs), K+, Na+, NH4 +, Cl-, and PO4 (-3), were separately introduced into clean, surgically sealed pouches. Studies were undertaken in 27 sheep, each with three pouches in the middle of the duodenum, jejunum, and ileum. Samples were taken at 15-minute intervals for 60 minutes to determine the absorption rates. The experimental results showed that L- and D-lactic acid were absorbed from the pouches of the duodenum, jejunum, and ileum throughout the 60 minutes. In the test solutions with pH 5.3, 420mOsmol/kg, and 12.5mM lactic acid that are in vivo conditions of light lactic acidosis, the mean absorption rates of D-lactic acid and L-lactic acid pooled from three pouches were similar, 0.07micro mol/cm2/min and 0.06micro mol/cm2/min, respectively, based on absorptive surface area. The mean absorption rates of DL-lactic acid from the duodenum, jejunum, and ileum pouches were almost the same, 0.14, 0.14, and 0.11micro mol/cm2/min, respectively. The absorption of lactic acid varied depending on lactic acid concentration, and there was a curvilinear relationship between lactic acid concentration and its absorption rate. A decrease in pH and osmotic pressure resulted in significant, corresponding increases in the absorption of lactic acid (P<0.0001 and P<0.05, respectively).     

Vaccinia virus H2 protein is an essential component of a complex involved in virus entry and cell-cell fusion

The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.     

Multiplicity of carriers for free glucalogues in hamster small intestine

The transport of free glucalogues across the mucosal pole of hamster small intestine is carried out by (at least) two transport systems, one of which is common to glucose, galactose, 6-deoxyglucose and 3-methylglucose, whereas the order is essentially specific for glucose and galactose, among the monosaccharides tested. This was shown by:

1. 1. The dependence of the unidirectional medium-mucosa flux on the substrate concentration which is described by a single horizontal hyperbola in the cases of 6-deoxyglucose and of 3-O-methylglucose, but by the sum of two such hyperbolas in the cases of both glucose and galactose.

2. 2. The mutual inhibition among glucalogues and the inhibition by arbutin, which are best described by the existence of two carriers, as given above:

3. 3. The difference in the absorption of glucose and galactose in baby hamsters as compared with adult hamsters.

Both systems are Na⁺-dependent and (to a different degree) phlorizin-sensitive. Neither is identical with the fructose transport system.     

Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainate excitotoxicity

Mutations in parkin, which encodes a RING domain protein associated with ubiquitin ligase activity, lead to autosomal recessive Parkinson's disease characterized by midbrain dopamine neuron loss. Here we show that parkin functions in a multiprotein ubiquitin ligase complex that includes the F-box/WD repeat protein hSel-10 and Cullin-1. HSel-10 serves to target the parkin ubiquitin ligase activity to cyclin E, an hSel-10-interacting protein previously implicated in the regulation of neuronal apoptosis. Consistent with the notion that cyclin E is a substrate of the parkin ubiquitin ligase complex, parkin deficiency potentiates the accumulation of cyclin E in cultured postmitotic neurons exposed to the glutamatergic excitotoxin kainate and promotes their apoptosis. Furthermore, parkin overexpression attenuates the accumulation of cyclin E in toxin-treated primary neurons, including midbrain dopamine neurons, and protects them from apoptosis.     

Development of substrate cycle enzymes in the hamster small intestine

A system of enzymes is required for the transport of reducing equivalents from reduced nicotinamide adenine dinucleotide (NADH) generated in the cytosol into the mitochondria by the substrate cycles. These substrate cycle enzymes are necessary for the flow of pyruvate derived from glucose into the mitochondria for oxidative decarboxylation and for the efficient production of adenosine 5′-triphosphate (ATP) for the unique intestinal nutrient transport functions. The enzymes of the l-glycerol 3-phosphate and the l-malate/l-aspartate substrate cycles are present before birth and increase significantly at the 7-day postnatal period of development. The key enzymes monitored in the intestinal subcellular fractions were NAD-linked l-glycerol-3-phosphate dehydrogenase, flavoprotein-linked l-glycerol-3-phosphate dehydrogenase, l-malate dehydrogenase, and l-glutamate-oxaloacetate transaminase.     

A growth-stimulating activity derived from the proximal small intestine is associated with an adaptive response

Luminal nutrition is important for the maintenance of small intestinal structure and function. The equilibrium between crypt cell production and villous cell loss in the mucosal epithelium of the small intestine is altered under certain conditions such as after a small bowel resection. Immediately after resection, there is a marked increase in crypt cell proliferation giving rise to an adaptive hyperplasia in the remnant intestine and for this response luminal nutrition is a critical factor. We have previously demonstrated the presence of a growth-stimulating (GS) activity in a heat-stable acidic extract of the rat proximal intestine 24, 48, and 96 h after resection, which is coincidental with an increase in crypt cell proliferation as measured by thymidine kinase activity. Eight days after resection when the GS activity is no longer detectable, the thymidine kinase activity returns to control values. The molecular weights of the peptides associated with this GS activity are 4500 and 1500, as determined by Sephadex gel filtration. Of note is that the oral intake of food is necessary for the appearance of the GS activity postoperatively. The presence of the GS activity has also been demonstrated upon refeeding after a fast, as well as at weaning in the rat, two physiological situations known to be associated with increased proliferation in the small intestine. This GS activity in the proximal intestine first detected in the resection model may represent a general mechanism by which food controls the cell renewal pattern of the small intestine.     

UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting

Endosomal sorting complexes required for transport (ESCRTs) regulate several events involving membrane invagination, including multivesicular body (MVB) biogenesis, viral budding, and cytokinesis. In each case, upstream ESCRTs combine with additional factors, such as Bro1 proteins, to recruit ESCRT-III and the ATPase VPS4 in order to drive membrane scission. A clue to understanding how such diverse cellular processes might be controlled independently of each other has been the identification of ESCRT isoforms. Mammalian ESCRT-I comprises TSG101, VPS28, VPS37A-D, and MVB12A/B. These could generate several ESCRT-I complexes, each targeted to a different compartment and able to recruit distinct ESCRT-III proteins. Here we identify a novel ESCRT-I component, ubiquitin-associated protein 1 (UBAP1), which contains a region conserved in MVB12. UBAP1 binds the endosomal Bro1 protein His domain protein tyrosine phosphatase (HDPTP), but not Alix, a Bro1 protein involved in cytokinesis. UBAP1 is required for sorting EGFR to the MVB and for endosomal ubiquitin homeostasis, but not for cytokinesis. UBAP1 is part of a complex that contains a fraction of total cellular TSG101 and that also contains VPS37A but not VPS37C. Hence, the presence of UBAP1, in combination with VPS37A, defines an endosome-specific ESCRT-I complex.     

Prion protein is a component of the multimolecular signaling complex involved in T cell activation

In this study we analyzed the interaction of prion protein PrP^C with components of glycosphingolipid-enriched microdomains in lymphoblastoid T cells. PrP^C was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrP^C is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrP^C and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP-70 was immunoprecipitated by anti-PrP after T cell activation. These results demonstrate that PrP^C interacts with ZAP-70, suggesting that PrP^C is a component of the multimolecular signaling complex within microdomains involved in T cell activation.     

Aubergine is a component of a nanos mRNA localization complex

Localization of nanos (nos) mRNA to the posterior pole of the Drosophila oocyte is essential for abdominal segmentation and germline development during embryogenesis. Posterior localization is mediated by a complex cis-acting localization signal in the nos 3' untranslated region that comprises multiple partially redundant elements. Genetic analysis suggests that this signal is recognized by RNA-binding proteins and associated factors that package nos mRNA into a localization competent ribonucleoprotein complex. However, functional redundancy among localization elements has made the identification of individual localization factors difficult. Indeed, only a single direct-acting nos localization factor, Rumpelstiltskin (Rump), has been identified thus far. Through a sensitized genetic screen, we have now identified the Argonaute family member Aubergine (Aub) as a nos localization factor. Aub interacts with nos mRNA in vivo and co-purifies with Rump in an RNA-dependent manner. Our results support a role for Aub, independent of its function in RNA silencing, as a component of a nos mRNA localization complex.