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ERD2, an essential yeast gene involved in protein sorting of luminal ER proteins
Cell Biology international
Reports, Vol. 14, Abstracts Supplement
A TRUNCATED FORM OF THE UROKINASE RECEPTOR WHICH IS RETAINED INTRACELLULARLY IN THE LJNGLYCOSYLATEDSTATE Lisbeth B. Meller, Francesco Blasi, University Institute of Microbiology, 0ster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark. The construction and expression of mutated membrane proteins provides a tool to interfere with the poorly known mechanisms by which these proteins are transported through the ER, to the golgi and subsequently expressed on the cell surface. The an important regulatory comurokinase receptor, ponent in plasminogen activation and extracellular is a membrane attached protein which proteolysis, In the present study, a is highly glycosylated. mutant, truncated form of the human urokinase reThis mutant ceptor was constructed and analysed. protein lacks the last 8 amino acid residues at the C terminus of the sequence encoded by the wild type cDNA. Mouse LB6 cells were transfected with the human mutant or wild type cDNA. Comparison revealed that the mutant transfectants, unlike the wild accumulated receptor activity type transfectants, ligand binding capability) in an intra(i.e. in addition to the cell surcellular compartment, face receptor which was expressed by both types. The truncation did not affect any potential site However, the intrafor N-bound glycosylation. cellular receptor variant proved to be completely In contrast, the suror almost unglycosylated. face expressed mutant receptor is as highly glycoThe results sylated as the wild type receptor. suggest that sequences in the very C-terminus of the protein affect the glycosylation processing and the transport towards the membrane, presumably by interference with the transport through the ER.
Wl
w3
REDUCING SECRETION
AGENTS OF IgM
INDUCE ASSEMBLY
Cristina Alberini*, Paola Bet”, Dario Finazzi*, Cesar Milstein+, Roberto Sitia”“. Universita di Brescia, Brescia, Italy*, Istituto Nazionale per la Ricerca sul Cancro, Genoa, It&^, MRC-Laboratory of Molecular Biology, Cambridge, UK+, Istituto San Raffaele, Milano, Italy”. A variety of editing devices are probably located along the secretory pathway, which allow only correctly assembled proteins to be transported to their final destination. For instance, B lymphocytes synthesize but do not secrete IgM, while only the polymeric form of IgM is secreted by plasma cells. Secretion of IgM by plasma cells is much less efficient than IgG and J chains are not secreted unless as part of IgM or IgA. All these processes can be selectively reverted by the addition of reducing agents to the culture medium, at concentrations which do not affect cell growth or cell viability. A role for free thiol groups in preventing the free flow through the secretory pathway is proposed whereby protein intermediates interact with resident proteins of the endoplasmic reticulum. Such interactions may be prevented by altering the intracellular redox potential or by site directed mutagenesis of the relevant cysteine residue(s). Supported by CNR and AIM!.
w2
1990
43
ERD2, an essential yeast gene involved in protein sorting of luminal ER proteins
Jan Sunewe, Kevin 0. Hardwick, Michael J. Lewis. Neta Dean and Hugh R.B. Pelham; MRC. LhfB. Hills Rd. Cambridge CB2 2QH, England The C-terminal signal HDBL is both necessary and sufficient for the retention of luminal ER proteins in S. cercvisiac. We have isolated mutants that fail to retain their endogenous ER proteins. They define two genes (ERDZ and ERD2) which have been cloned and sequenced. Here we describe ERD2 an iutron-wnteining gene encoding a 26 kDa membrane protein. Disruption of one chromosomal copy in a diploid strain produced a recessive lethal mutation. To vuify this essential function a haploid strain containing this null allele and ERDZ under CAL control was constructed. Repression of the CAL promoter with glucose arrested growth after J2h and led to the secretion of endogenous ER proteins. Electronmicrographs of this conditional mutant showed an increase in the amount of BR membranes. Analysis of the wt protein sequence revealed an internal repeat of 18 aa of which 14 aa are conserved or analogous. These two repeats score as amphipathic helices in a computer analysis. Defining the regions involved in HDBL retention three of the original UV-generated alleles of erdZ were cloned by PCR. One showed a mutation in this repeat.The changefrom Asp to Asn eliminatesa negative charge and has a dramatic effect on HDBL retention. The two other mutations are less subtle changes that may influence the folding of the protein. From this data and pulse chase experiments we conclude that ERDZ has an important role in HDBL retention . Its molecular function in direct HDEL recognition andfor membrane traffic has to be established. Supported by Roche Research Foundation.
TUNICAMYCIN-INDUCIBLE LOOKING FOR PROTEINS 1N PLANT CELLS Alessandro Vitale, M. Serena Fabbrini, Robert;0 Bollini and Aldo Ceriotti. Istituo Biosintesi Vegetali, CNR, Milano, Italy. conditions, stress Under a variety of including tunicamycin (Tm) treatment, animal cells can accumulate malfolded proteins in the reticulum (ER). This induces a endoplasmic dramatic increase in the synthesis of at least GRP78 and GRP94, which two ER-resident proteins, are believed to have the function of preventing or disrupting inappropriate interactions of newly synthesized proteins (reviewed by Deshaies et al, TIBS 13, 384, 1988). During seed development, bean cotyledonary cells synthesize large amounts phaseolin, of the vacuolar glycoproteins phytohemagglutinin and alfa-amylase inhibitor. We report that, although Tm treatment does not prevent exit of the unglycosylated forms of these proteins from the Golgi-ER system, it greatly enhances the synthesis of at least one protein, having Mr around 80 Kd. This protein is resident of the ER-Golgi system. We also identified an abundant glycoprorein resident of the ER-Golgi system, having Mr around 75Kd. Under Tm treatment, the unglycosylated form of this protein appears to have greatly enhanced turnover.
w4
Reports, Vol. 14, Abstracts Supplement
A TRUNCATED FORM OF THE UROKINASE RECEPTOR WHICH IS RETAINED INTRACELLULARLY IN THE LJNGLYCOSYLATEDSTATE Lisbeth B. Meller, Francesco Blasi, University Institute of Microbiology, 0ster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark. The construction and expression of mutated membrane proteins provides a tool to interfere with the poorly known mechanisms by which these proteins are transported through the ER, to the golgi and subsequently expressed on the cell surface. The an important regulatory comurokinase receptor, ponent in plasminogen activation and extracellular is a membrane attached protein which proteolysis, In the present study, a is highly glycosylated. mutant, truncated form of the human urokinase reThis mutant ceptor was constructed and analysed. protein lacks the last 8 amino acid residues at the C terminus of the sequence encoded by the wild type cDNA. Mouse LB6 cells were transfected with the human mutant or wild type cDNA. Comparison revealed that the mutant transfectants, unlike the wild accumulated receptor activity type transfectants, ligand binding capability) in an intra(i.e. in addition to the cell surcellular compartment, face receptor which was expressed by both types. The truncation did not affect any potential site However, the intrafor N-bound glycosylation. cellular receptor variant proved to be completely In contrast, the suror almost unglycosylated. face expressed mutant receptor is as highly glycoThe results sylated as the wild type receptor. suggest that sequences in the very C-terminus of the protein affect the glycosylation processing and the transport towards the membrane, presumably by interference with the transport through the ER.
Wl
w3
REDUCING SECRETION
AGENTS OF IgM
INDUCE ASSEMBLY
Cristina Alberini*, Paola Bet”, Dario Finazzi*, Cesar Milstein+, Roberto Sitia”“. Universita di Brescia, Brescia, Italy*, Istituto Nazionale per la Ricerca sul Cancro, Genoa, It&^, MRC-Laboratory of Molecular Biology, Cambridge, UK+, Istituto San Raffaele, Milano, Italy”. A variety of editing devices are probably located along the secretory pathway, which allow only correctly assembled proteins to be transported to their final destination. For instance, B lymphocytes synthesize but do not secrete IgM, while only the polymeric form of IgM is secreted by plasma cells. Secretion of IgM by plasma cells is much less efficient than IgG and J chains are not secreted unless as part of IgM or IgA. All these processes can be selectively reverted by the addition of reducing agents to the culture medium, at concentrations which do not affect cell growth or cell viability. A role for free thiol groups in preventing the free flow through the secretory pathway is proposed whereby protein intermediates interact with resident proteins of the endoplasmic reticulum. Such interactions may be prevented by altering the intracellular redox potential or by site directed mutagenesis of the relevant cysteine residue(s). Supported by CNR and AIM!.
w2
1990
43
ERD2, an essential yeast gene involved in protein sorting of luminal ER proteins
Jan Sunewe, Kevin 0. Hardwick, Michael J. Lewis. Neta Dean and Hugh R.B. Pelham; MRC. LhfB. Hills Rd. Cambridge CB2 2QH, England The C-terminal signal HDBL is both necessary and sufficient for the retention of luminal ER proteins in S. cercvisiac. We have isolated mutants that fail to retain their endogenous ER proteins. They define two genes (ERDZ and ERD2) which have been cloned and sequenced. Here we describe ERD2 an iutron-wnteining gene encoding a 26 kDa membrane protein. Disruption of one chromosomal copy in a diploid strain produced a recessive lethal mutation. To vuify this essential function a haploid strain containing this null allele and ERDZ under CAL control was constructed. Repression of the CAL promoter with glucose arrested growth after J2h and led to the secretion of endogenous ER proteins. Electronmicrographs of this conditional mutant showed an increase in the amount of BR membranes. Analysis of the wt protein sequence revealed an internal repeat of 18 aa of which 14 aa are conserved or analogous. These two repeats score as amphipathic helices in a computer analysis. Defining the regions involved in HDBL retention three of the original UV-generated alleles of erdZ were cloned by PCR. One showed a mutation in this repeat.The changefrom Asp to Asn eliminatesa negative charge and has a dramatic effect on HDBL retention. The two other mutations are less subtle changes that may influence the folding of the protein. From this data and pulse chase experiments we conclude that ERDZ has an important role in HDBL retention . Its molecular function in direct HDEL recognition andfor membrane traffic has to be established. Supported by Roche Research Foundation.
TUNICAMYCIN-INDUCIBLE LOOKING FOR PROTEINS 1N PLANT CELLS Alessandro Vitale, M. Serena Fabbrini, Robert;0 Bollini and Aldo Ceriotti. Istituo Biosintesi Vegetali, CNR, Milano, Italy. conditions, stress Under a variety of including tunicamycin (Tm) treatment, animal cells can accumulate malfolded proteins in the reticulum (ER). This induces a endoplasmic dramatic increase in the synthesis of at least GRP78 and GRP94, which two ER-resident proteins, are believed to have the function of preventing or disrupting inappropriate interactions of newly synthesized proteins (reviewed by Deshaies et al, TIBS 13, 384, 1988). During seed development, bean cotyledonary cells synthesize large amounts phaseolin, of the vacuolar glycoproteins phytohemagglutinin and alfa-amylase inhibitor. We report that, although Tm treatment does not prevent exit of the unglycosylated forms of these proteins from the Golgi-ER system, it greatly enhances the synthesis of at least one protein, having Mr around 80 Kd. This protein is resident of the ER-Golgi system. We also identified an abundant glycoprorein resident of the ER-Golgi system, having Mr around 75Kd. Under Tm treatment, the unglycosylated form of this protein appears to have greatly enhanced turnover.
w4