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H P Hauri

Publications and source records attributed to H P Hauri.

At least 37 records · Page 2Linked to original sources
PubMed1997

Sequence and overexpression of GPP130/GIMPc: evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein.

It is thought that residents of the Golgi stack are localized by a retention mechanism that prevents their forward progress. Nevertheless, some early Golgi proteins acquire late Golgi modifications. Herein, we describe GPP130 (Golgi phosphoprotein of 130 kDa), a 130-kDa phosphorylated and glycosylated integral membrane protein localized to the cis/medial Golgi. GPP130 appears to be the human counterpart of rat Golgi integral membrane protein, cis (GIMPc), a previously identified early Golgi antigen that acquires late Golgi carbohydrate modifications. The sequence of cDNAs encoding GPP130 indicate that it is a type II membrane protein with a predicted molecular weight of 81,880 and an unusually acidic lumenal domain. On the basis of the alignment with several rod-shaped proteins and the presence of multiple predicted coiled-coil regions, GPP130 may form a flexible rod in the Golgi lumen. In contrast to the behavior of previously studied type II Golgi proteins, overexpression of GPP130 led to a pronounced accumulation in endocytotic vesicles, and endogenous GPP130 reversibly redistributed to endocytotic vesicles after chloroquine treatment. Thus, localization of GPP130 to the early Golgi involves steps that are saturable and sensitive to lumenal pH, and GPP130 contains targeting information that specifies its return to the Golgi after chloroquine washout. Given that GIMPc acquires late Golgi modifications in untreated cells, it seems likely that GPP130/GIMPc continuously cycles between the early Golgi and distal compartments and that an unidentified retrieval mechanism is important for its targeting.

Amino Acid Sequence↗
PubMed1997

A mutation in a highly conserved region in brush-border sucrase-isomaltase and lysosomal alpha-glucosidase results in Golgi retention.

A point mutation in the cDNA of human intestinal sucrase-isomaltase has been recently identified in phenotype II of congenital sucrase-isomaltase deficiency. The mutation results in a substitution of glutamine by proline at position 1098 (Q1098P) in the sucrase subunit. Expression of this mutant sucrase-isomaltase cDNA in COS-1 cells results in an accumulation of sucrase-isomaltase in the ER, intermediate compartment and the cis-Golgi cisternae similar to the accumulation in phenotype II intestinal cells. An interesting feature of the Q1098P substitution is its location in a region of the sucrase subunit that shares striking similarities with the isomaltase subunit and other functionally related enzymes, such as human lysosomal acid alpha-glucosidase and Schwanniomyces occidentalis glucoamylase. We speculated that the Q-->P substitution in these highly conserved regions may result in a comparable accumulation. Here we examined this hypothesis using lysosomal alpha-glucosidase as a reporter gene. Mutagenesis of the glutamine residue at position 244 in the homologous region of alpha-glucosidase to proline results in a protein that is neither transported to the lysosomes nor secreted extracellularly but accumulates in the ER, intermediate compartment and cis-Golgi as a mannose-rich polypeptide similar to mutant sucrase-isomaltase in phenotype II. We propose that the Q1098P and Q244P mutations (in sucrase-isomaltase and alpha-glucosidase, respectively) generate structural alterations that are recognized by a control mechanism, operating beyond the ER in the intermediate compartment or cis-Golgi.

Amino Acid Sequence↗
PubMed1996

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

Based on sequence homologies with leguminous lectins, the intermediate compartment marker ERGIC-53 was proposed to be a member of a putative new class of animal lectins associated with the secretory pathway. Independent, a promyelocytic protein, MR60, was purified by mannose-column chromatography, and a cDNA was isolated that matched MR60 peptide sequences. This cDNA was identical to that of ERGIC-53 and homologies with the animal lectin family of the galectins were noticed. Not all peptide sequences of MR60, however, were found in ERGIC-53, raising the possibility that another protein associated with ERGIC-53 may possess the lectin activity. Here, we provide the first direct evidence for a lectin function of ERGIC-53. Overexpressed ERGIC-53 binds to a mannose column in a calcium-dependent manner and also co-stains with mannosylated neoglycoprotein in a morphological binding assay. By using a sequential elution protocol we show that ERGIC-53 has selectivity for mannose and low affinity for glucose and GlcNAc, but no affinity for galactose. To experimentally address the putative homology of ERGIC-53 to leguminous lectins, a highly conserved protein family with an invariant asparagine essential for carbohydrate binding, we substituted the corresponding asparagine in ERGIC-53. This mutation, as well as a mutation affecting a second site in the putative carbohydrate recognition domain, abolished mannose-column binding and co-staining with mannosylated neoglycoprotein. These findings establish ERGIC-53 as a lectin and provide functional evidence for its relationship to leguminous lectins. Based on its monosaccharide specificity, domain organization, and recycling properties, we propose ERGIC-53 to function as a sorting receptor for glyco-proteins in the early secretory pathway.

Amino Acid Sequence↗
PubMed1995

A C-terminally-anchored Golgi protein is inserted into the endoplasmic reticulum and then transported to the Golgi apparatus.

Unlike conventional membrane proteins of the secretory pathway, proteins anchored to the cytoplasmic surface of membranes by hydrophobic sequences near their C termini follow a posttranslational, signal recognition particle-independent insertion pathway. Many such C-terminally-anchored proteins have restricted intracellular locations, but it is not known whether these proteins are targeted directly to the membranes in which they will ultimately reside. Here we have analyzed the intracellular sorting of the Golgi protein giantin, which consists of a rod-shaped 376-kDa cytoplasmic domain followed by a hydrophobic C-terminal anchor sequence. Unexpectedly, we find that giantin behaves like a conventional secretory protein in that it inserts into the endoplasmic reticulum (ER) and then is transported to the Golgi. A deletion mutant lacking a portion of the cytoplasmic domain adjacent to the membrane anchor still inserts into the ER but fails to reach the Golgi, even though this mutant has a stable folded structure. These findings suggest that the localization of a C-terminally-anchored Golgi protein involves at least three steps: insertion into the ER membrane, controlled incorporation into transport vesicles, and retention within the Golgi.

Amino Acid Sequence↗
PubMed1995

A novel endocytosis signal related to the KKXX ER-retrieval signal.

Membrane proteins often contain a sorting signal in their cytoplasmic tail that promotes their clustering into coated vesicles at a specific cellular site. ERGIC-53 contains a cytoplasmic ER-retrieval signal, KKFF. However, overexpressed ERGIC-53 is transported to the cell surface and rapidly endocytosed. Here we report that ERGIC-53 carries a previously undescribed endocytosis signal. Surprisingly, the signal was KKFF and like the ER-retrieval signal required a C-terminal position. In fact, the minimal consensus sequence determined by substitutional mutagenesis (K-K/R-F/Y-F/Y) was related to the ER-retrieval consensus (K-K-X-X). Furthermore, we provide evidence that internalization of VIP36, a protein that cycles between plasma membrane and Golgi, is mediated by a signal at its C-terminus that matches the internalization consensus sequence. The relatedness of the two signals suggests that coatomer-mediated retrieval of proteins may be mechanistically more related to clathrin-dependent sorting than previously anticipated.

Amino Acid Sequence↗
PubMed1995

Selective reentry of recycling cell surface glycoproteins to the biosynthetic pathway in human hepatocarcinoma HepG2 cells.

Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans-Golgi network (TGN), the trans/medial- and cis-Golgi. Transport to these sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling these proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface. This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these compartments during biosynthesis en route to the surface. Reentry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. Return to the trans-Golgi was traced measuring the transfer of [3H]fucose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically labeled in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM). As a result surface proteins retained N-glycans of the oligomannosidic type. Return to the site of mannosidase I in the medial/cis-Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM. Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to the trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indicating that the early secretory pathway is not or is only at insignificant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did not return to the TGN or to other biosynthetic compartments in detectable amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling.

Animals↗
PubMed1995

Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway.

ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. To identify the targeting signals that mediate this recycling, N-glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER-ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC-cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC-53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER-retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant.

Amino Acid Sequence↗
PubMed1995

Segregation of ERGIC53 and the mammalian KDEL receptor upon exit from the 15 degrees C compartment.

Protein trafficking along the exocytotic pathway occurs by vesicular transport between successive membranous compartments. Transport from the endoplasmic reticulum (ER) to the Golgi apparatus has been proposed to be bridged by a morphologically defined ER-Golgi intermediate compartment (ERGIC). Using the subcellular dynamics of two markers for the ERGIC, the 53 kDa protein ERGIC53 and the mammalian KDEL receptor (KDEL-R), we have investigated the biochemical and physiological characteristics of ER-Golgi anterograde and retrograde transport. The KDEL-R at steady state is mainly confined to the perinuclear Golgi region while the ERGIC53 has a more elaborate distribution, including the ER. Both proteins can be colocalized to spotty structures distributed throughout the cytoplasm by incubating the cells at 15 degrees C. Upon returning the cells to 37 degrees C, the direction of transport for the two proteins diverged. KDEL-R was seen to emanate into tubular structures which eventually culminated in a focused, perinuclear staining. These dynamic changes are consistent with the anterograde transport process from the ER to the Golgi apparatus. ERGIC53, on the other hand, was distributed into an extended reticular network as well as the nuclear envelope, a staining pattern characteristic of the ER. With time, ERGIC53 was seen to return to the spotty structures again. The ER retrieval of ERGIC53 is consistent with the fact that the protein contains a dilysine motif which may function as an ER retrieval signal. The movement of ERGIC53 into the ER is not affected by microtubule disrupting agents, which inhibit the movement of KDEL-R to the Golgi. Both the processes are, however, sensitive to the alkylating agent N-ethylmaleimide. When reconstituted in vitro using digitonin permeabilized cells, the movement of ERGIC53 into the ER has a requirement for metabolic energy, is partially inhibited by the nonhydrolyzable guanine nucleotide analog GTP gamma S but could not be made to be cytosol dependent. These results documented the convergence of anterograde transport and retrograde transport at the 15 degrees C compartment and implied the existence of a segregation or a sorting process that would result in the segregation of proteins with different targeting signals in the structure.

Animals↗
PubMed1994

A dual role for COOH-terminal lysine residues in pre-Golgi retention and endocytosis of ERGIC-53.

ERGIC-53 (former designation, p53) is a 53-kDa nonglycosylated, dimeric, and hexameric type I membrane protein that has been established as a marker protein for a tubulovesicular intermediate compartment in which protein transport from the endoplasmic reticulum to the Golgi apparatus is blocked at 15 degrees C. Although ERGIC-53 is not a resident protein of the rough endoplasmic reticulum its cDNA sequence carries a double lysine endoplasmic reticulum retention motif at the cytoplasmically exposed COOH terminus. Here we report that overexpression of ERGIC-53 in COS cells saturates its intracellular retention system leading to the appearance of ERGIC-53 at the cell surface. Cell surface ERGIC-53 is efficiently endocytosed by a mechanism that is disturbed when the two critical lysines of the endoplasmic reticulum retention motif are replaced by serines. The results suggest a mechanistic similarity of pre-Golgi retention by the double lysine motif and lysine-based endocytosis.

Amino Acid Sequence↗
PubMed1994

Localization of O-glycan initiation, sphingomyelin synthesis, and glucosylceramide synthesis in Vero cells with respect to the endoplasmic reticulum-Golgi intermediate compartment.

The identification of an endoplasmic reticulum-Golgi intermediate compartment (ERGIC), defined by the 53-kDa transmembrane marker protein ERGIC-53, has added to the complexity of the exocytic pathway of higher eukaryotic cells. Recently, a subcellular fractionation procedure was established for the isolation of the ERGIC from Vero cells (Schweizer, A., Matter, K., Ketcham, C. M., and Hauri, H.-P. (1991) J. Cell Biol. 113, 45-54) which provides a means to study more precisely the compartmentalization of the various enzymic functions along the early secretory pathway. Here, we have investigated if O-glycan initiation and sphingomyelin synthesis are associated with the ERGIC by analyzing both the responsible enzyme activities and their corresponding products. Moreover, the synthesis of glucosylceramide, the precursor of most glycosphingolipids, was also analyzed. In the purified ERGIC fraction UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc transferase) was only minimally enriched, sphingomyelin synthase was not enriched, and UDP-glucose:ceramide-glucosyl transferase specific activity was lower than in the homogenate. On Percoll gradients all three enzymes cofractionated with Golgi markers rather than ERGIC-53. Accordingly, sphingomyelin concentrations were extremely low in the ERGIC fraction. Double immunofluorescence localization of core N-acetylgalactosamine, the product of GalNAc transferase, by monoclonal antibodies against GalNAc-Ser/Thr (Tn antigen) revealed only little apparent overlap with ERGIC-53. This was particularly evident in brefeldin A-treated cells which showed entirely different patterns of Tn antigens and ERGIC-53. The results suggest that in the secretory pathway of Vero cells O-glycan initiation and sphingomyelin as well as glucosylceramide synthesis mainly occur beyond the ERGIC in the Golgi apparatus.

Amino Acid Sequence↗
PubMed1994

Induction of lactase biosynthesis in the human intestinal epithelial cell line Caco-2.

The human colonic adenocarcinoma cell line Caco-2 forms monolayers of differentiated enterocyte-like cells when cultured on permeable supports. After confluency, Caco-2 cells express a number of brush-border enzymes including lactase-phlorizin hydrolase, sucrase-isomaltase and dipeptidylpeptidase IV. We have studied, with particular emphasis on lactase-phlorizin hydrolase, the modulation of biosynthesis of these enzymes by stimulating second messenger systems. Forskolin induced lactase-phlorizin hydrolase synthesis approximately fourfold within 7 h, suppressed sucrase-isomaltase synthesis, and had little effect on dipeptidylpeptidase IV. Dibutyryl-cAMP, 8-bromo-cAMP and vasoactive intestinal peptide also increased lactase-phlorizin hydrolase biosynthesis, indicating c-AMP dependent regulation. The induction of lactase-phlorizin hydrolase biosynthesis could be inhibited by actinomycin D and was preceded by a fourfold increase in lactase-phlorizin hydrolase mRNA levels, suggesting transcriptional control. Phorbol 12-myristate 13-acetate had an inhibitory effect on brush-border enzyme synthesis, in particular on sucrase-isomaltase, and blocked the forskolin-induced biosynthesis of lactase-phlorizin hydrolase. Lactase-phlorizin hydrolase synthesis was also inducible by hydrocortisone, but maximal induction required at least 3 days during which time sucrase-isomaltase synthesis diminished. The results indicate opposite regulation of lactase-phlorizin hydrolase and sucrase-isomaltase via cAMP and corticosteroids, and suggest that the Caco-2 cell line can serve as a model system to study aspects of the humoral regulation of human intestinal brush-border enzymes in cell culture.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Retention of p63 in an ER-Golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers.

The type II membrane protein p63 is a resident protein of a membrane network interposed between rough ER and Golgi apparatus. To study the retention of p63, mutant forms were expressed in COS cells and the intracellular distribution determined by immunofluorescence microscopy. Investigation of chimeric constructs between p63 and the plasma membrane protein dipeptidylpeptidase IV showed that protein sequences from all three domains of the p63 protein are required to achieve complete intracellular retention. Mutational analysis of the 106-amino acid cytoplasmic tail of p63 revealed that the NH2-terminal 23 amino acids are necessary for retention. When p63 was solubilized with Triton X-100 and subjected to centrifugation at 100,000 g, it formed large, insoluble oligomers, particularly at neutral pH and below. A comparison of the behavior of wildtype and mutant p63 proteins in this assay revealed a perfect correlation between the formation of large oligomers and correct intracellular retention. These results suggest that self-association may be a major mechanism by which p63 is retained between the rough ER and the Golgi apparatus.

Amino Acid Sequence↗
PubMed1994

Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit.

During the formation of the 3' ends of mRNA, the cleavage and polyadenylation specificity factor (CPSF) is required for 3' cleavage of the transcript as well as for subsequent polyadenylation. Using peptide sequences from a tryptic digest, we have cloned the 100-kDa subunit of CPSF. This subunit is a novel protein showing no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and monoclonal antibodies were used to characterize the composition of CPSF. Immunoprecipitations of CPSF from HeLa cell extracts and from labeled chromatographic fractions show the coprecipitation of all four subunits of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are specifically cross-linked to precursor RNA by UV irradiation were identified as CPSF subunits by immunoprecipitation. Immunofluorescent detection of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.

Amino Acid Sequence↗
PubMed1993

Separation of splicing factor SF3 into two components and purification of SF3a activity.

Components required for the splicing of nuclear messenger RNA precursors in vitro have been isolated from HeLa cells. Here we describe the separation of splicing factor SF3 into two components, SF3a and SF3b. Both activities are required together with several other protein factors and U1 and U2 small nuclear ribonucleoproteins for the assembly of a presplicing complex which represents the first ATP-dependent step in the assembly of the active spliceosome. SF3a has been purified to homogeneity by a combination of ion-exchange chromatography, gel filtration, and glycerol gradient sedimentation. It consists of a complex of three polypeptides of 60, 66, and 120 kDa. The association of SF3a activity with these polypeptides has been confirmed by immunoprecipitation and depletion experiments using a monoclonal antibody directed against the 66-kDa subunit.

Blotting, Western↗
PubMed1993

Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo.

Microinjection of antibodies against a synthetic peptide of a non-clathrin-coated vesicle-associated coat protein, beta-COP, blocks transport of a temperature-sensitive vesicular stomatitis virus glycoprotein (ts-O45-G) to the cell surface. Transport is inhibited upon release of the viral glycoprotein from temperature blocks at 39.5 degrees C (endoplasmic reticulum [ER]) and 15 degrees C (intermediate compartment), but not at 20 degrees C (trans-Golgi network). Ts-O45-G is arrested in tubular membrane structures containing p53 at the interface of the ER and the Golgi stack. This is consistent with inhibition of acquisition of endoglycosidase H resistance of ts-O45-G in injected cells. Secretion of endogenous proteins and maturation of cathepsin D are also inhibited. These data provide in vivo evidence that beta-COP has an important function in biosynthetic membrane traffic in mammalian cells.

Animals↗
PubMed1993

Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa.

The Golgi complex consists of a series of stacked cisternae in most eukaryotes. Morphological studies indicate the existence of intercisternal cross-bridge structures that may mediate stacking, but their identity is unknown. We have identified a 400-kDa protein, giantin, that is localized to the Golgi complex because its staining in double immunofluorescence experiments was coincident with that of galactosyltransferase, both in untreated cells and in cells treated with agents that disrupt Golgi structure. A monoclonal antibody against giantin yielded Golgi staining in one avian and all mammalian cell types tested, indicating that giantin is a conserved protein. Giantin exhibited reduced mobility on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was recovered in membrane fractions after differential centrifugation or sucrose flotation, and was not released from membranes by carbonate extraction. Thus, giantin appears to be an integral component of the Golgi membrane with a disulfide-linked lumenal domain. Strikingly, the majority of the polypeptide chain is cytoplasmically disposed, because large (up to 350 kDa) proteolytic fragments of giantin could be released from intact Golgi vesicles. This feature, a large contiguous cytoplasmic domain, is present in the calcium-release channel of muscle that cross-bridges the sarcoplasmic reticulum and transverse tubule membranes. Therefore, giantin's localization, conservation, and physical properties suggest that it may participate in forming the intercisternal cross-bridges of the Golgi complex.

Adenocarcinoma↗
PubMed1993

The constitutive exocytotic pathway in microvillous atrophy.

Microvillous atrophy is a disorder within the intractable diarrhea of infancy syndrome. The disease is believed to stem from a transport defect that prevents exocytosis of brush border-related material. We investigated this hypothesis by examining the direct constitutive exocytotic pathway using sucrase-isomaltase as a representative protein. We also studied various other brush border and lysosomal marker enzymes. The biosynthesis and localization of selected intestinal epithelial enzymes were studied in small-intestinal mucosal biopsy specimens from a total of nine children with microvillous atrophy by: (a) metabolic labeling in organ culture, (b) radioiodination and immunoprecipitation, (c) indirect immunoperoxidase immunocytochemistry, and (d) immunogold electron microscopy. The results demonstrated that brush border enzymes were synthesized normally and could be located in the apical brush border membrane and on microvillous membrane within microvillous inclusions. Brush border enzymes were not detected in the "secretory granules" that accumulated within the apical cytoplasm of epithelial cells. Lysosomal enzymes were only detected within lysosomal bodies. Thus, the direct constitutive pathway is not involved in microvillous atrophy, and a disturbance of endocytosis or the indirect constitutive pathway is unlikely. Any transport defect in the disease probably involves a different, unidentified exocytotic pathway.

Atrophy↗