Yeast genes controlling responses to topogenic signals in a model transmembrane protein.

Yeast protein insertion orientation (PIO) mutants were isolated by selecting for growth on sucrose in cells in which the only source of invertase is a C-terminal fusion to a transmembrane protein. Only the fraction with an exocellular C terminus can be processed to secreted invertase and this fraction is constrained to 2-3% by a strong charge difference signal. Identified pio mutants increased this to 9-12%. PIO1 is SPF1, encoding a P-type ATPase located in the endoplasmic reticulum (ER) or Golgi. spf1-null mutants are modestly sensitive to EGTA. Sensitivity is considerably greater in an spf1 pmr1 double mutant, although PIO is not further disturbed. Pmr1p is the Golgi Ca(2+) ATPase and Spf1p may be the equivalent ER pump. PIO2 is STE24, a metalloprotease anchored in the ER membrane. Like Spf1p, Ste24p is expressed in all yeast cell types and belongs to a highly conserved protein family. The effects of ste24- and spf1-null mutations on invertase secretion are additive, cell generation time is increased 60%, and cells become sensitive to cold and to heat shock. Ste24p and Rce1p cleave the C-AAX bond of farnesylated CAAX box proteins. The closest paralog of SPF1 is YOR291w. Neither rce1-null nor yor291w-null mutations affected PIO or the phenotype of spf1- or ste24-null mutants. Mutations in PIO3 (unidentified) cause a weaker Pio phenotype, enhanced by a null mutation in BMH1, one of two yeast 14-3-3 proteins.

[1]  P. Espenshade,et al.  Regulated Step in Cholesterol Feedback Localized to Budding of SCAP from ER Membranes , 2000, Cell.

[2]  D. Tipper,et al.  Use of β‐lactamase as a secreted reporter of promoter function in yeast , 1994 .

[3]  B. Chaudhuri,et al.  A modified Kex2 enzyme retained in the endoplasmic reticulum prevents disulfide‐linked dimerisation of recombinant human insulin‐like growth factor‐1 secreted from yeast , 1992, FEBS letters.

[4]  R. Dalbey,et al.  The Proton Motive Force, Acting on Acidic Residues, Promotes Translocation of Amino-terminal Domains of Membrane Proteins When the Hydrophobicity of the Translocation Signal Is Low* , 1998, The Journal of Biological Chemistry.

[5]  S. Michaelis,et al.  Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Y. Shimma,et al.  P‐type ATPase spf1 mutants show a novel resistance mechanism for the killer toxin SMKT , 1999, Molecular microbiology.

[7]  T. Rapoport,et al.  The Sec61p Complex Mediates the Integration of a Membrane Protein by Allowing Lipid Partitioning of the Transmembrane Domain , 2000, Cell.

[8]  K. Redding,et al.  Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae , 1991, The Journal of cell biology.

[9]  G. Fink,et al.  The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family , 1989, Cell.

[10]  B. Kruijff,et al.  The Role of Anionic Lipids in Protein Insertion and Translocation in Bacterial Membranes , 1998, The Journal of Membrane Biology.

[11]  R. Plemper,et al.  The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. , 1998, Molecular biology of the cell.

[12]  D Botstein,et al.  Functional Analysis of the Genes of Yeast Chromosome V by Genetic Footprinting , 1996, Science.

[13]  A. Myers,et al.  Yeast/E. coli shuttle vectors with multiple unique restriction sites , 1986, Yeast.

[14]  G von Heijne,et al.  Anionic phospholipids are determinants of membrane protein topology , 1997, The EMBO journal.

[15]  G. von Heijne,et al.  N-tail translocation in a eukaryotic polytopic membrane protein: synergy between neighboring transmembrane segments. , 1999, European journal of biochemistry.

[16]  J Vandekerckhove,et al.  A Plant Plasma Membrane H+-ATPase Expressed in Yeast Is Activated by Phosphorylation at Its Penultimate Residue and Binding of 14-3-3 Regulatory Proteins in the Absence of Fusicoccin* , 2000, The Journal of Biological Chemistry.

[17]  P. Philippsen,et al.  Heterologous HIS3 Marker and GFP Reporter Modules for PCR‐Targeting in Saccharomyces cerevisiae , 1997, Yeast.

[18]  D. Tipper,et al.  Transmembrane Protein Insertion Orientation in Yeast Depends on the Charge Difference across Transmembrane Segments, Their Total Hydrophobicity, and Its Distribution* , 1998, The Journal of Biological Chemistry.

[19]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[20]  Martin Spiess,et al.  Multiple Determinants Direct the Orientation of Signal–Anchor Proteins: The Topogenic Role of the Hydrophobic Signal Domain , 1997, The Journal of cell biology.

[21]  A. Goffeau,et al.  The complete inventory of the yeast Saccharomyces cerevisiae P‐type transport ATPases , 1997, FEBS letters.

[22]  K. Axelsen,et al.  Evolution of Substrate Specificities in the P-Type ATPase Superfamily , 1998, Journal of Molecular Evolution.

[23]  J. Beltzer,et al.  Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence. , 1991, The Journal of biological chemistry.

[24]  David Botstein,et al.  Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase , 1982, Cell.

[25]  R. Hampton,et al.  Regulation of Hmg-Coa Reductase Degradation Requires the P-Type Atpase Cod1p/Spf1p , 2000, The Journal of cell biology.

[26]  Y. S. Zhu,et al.  Use of beta-lactamase as a secreted reporter of promoter function in yeast. , 1994, Yeast.

[27]  D. Tipper,et al.  The Role of Charged Residues in Determining Transmembrane Protein Insertion Orientation in Yeast* , 1996, The Journal of Biological Chemistry.

[28]  J. Rine,et al.  Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor. , 1998, Genetics.

[29]  T A Rapoport,et al.  Predicting the orientation of eukaryotic membrane-spanning proteins. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[30]  G. Fink,et al.  14-3-3 Proteins Are Essential for RAS/MAPK Cascade Signaling during Pseudohyphal Development in S. cerevisiae , 1997, Cell.

[31]  Heijne,et al.  Membrane protein topology: effects of delta mu H+ on the translocation of charged residues explain the ‘positive inside’ rule. , 1994, The EMBO journal.

[32]  J. Rine,et al.  The CaaX Proteases, Afc1p and Rce1p, Have Overlapping but Distinct Substrate Specificities , 2000, Molecular and Cellular Biology.

[33]  V. Goder,et al.  Glycosylation Can Influence Topogenesis of Membrane Proteins and Reveals Dynamic Reorientation of Nascent Polypeptides within the Translocon , 1999, The Journal of cell biology.

[34]  J. Beckwith,et al.  The Protein Translocation Apparatus Contributes to Determining the Topology of an Integral Membrane Protein in Escherichia coli * , 1998, The Journal of Biological Chemistry.

[35]  C. Suzuki.  Immunochemical and Mutational Analyses of P-type ATPase Spf1p Involved in the Yeast Secretory Pathway , 2001, Bioscience, biotechnology, and biochemistry.

[36]  L. Lehle,et al.  Ca(2+)-ATPases of Saccharomyces cerevisiae: diversity and possible role in protein sorting. , 1998, FEMS microbiology letters.

[37]  Jonathan W. Yewdell,et al.  Rapid degradation of a large fraction of newly synthesized proteins by proteasomes , 2000, Nature.

[38]  D. Tipper,et al.  Yeast dsRNA viruses: replication and killer phenotypes , 1991, Molecular microbiology.

[39]  G. Heijne.  Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. , 1992, Journal of molecular biology.

[40]  G von Heijne,et al.  Topological Rules for Membrane Protein Assembly in Eukaryotic Cells* , 1997, The Journal of Biological Chemistry.

[41]  Gunnar von Heijne,et al.  Fine-tuning the topology of a polytopic membrane protein: Role of positively and negatively charged amino acids , 1990, Cell.

[42]  S. Michaelis,et al.  A Novel Membrane-associated Metalloprotease, Ste24p, Is Required for the First Step of NH2-terminal Processing of the Yeast a-Factor Precursor , 1997, The Journal of cell biology.

[43]  R. Hegde,et al.  Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[44]  R. Schlegel,et al.  A Subfamily of P-Type ATPases with Aminophospholipid Transporting Activity , 1996, Science.

[45]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[46]  H. Komano,et al.  Shared functions in vivo of a glycosyl-phosphatidylinositol-linked aspartyl protease, Mkc7, and the proprotein processing protease Kex2 in yeast. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[47]  H. Ulrich,et al.  Kex2‐dependent invertase secretion as a tool to study the targeting of transmembrane proteins which are involved in ER‐‐>Golgi transport in yeast. , 1994, The EMBO journal.

[48]  Peter Walter,et al.  Functional and Genomic Analyses Reveal an Essential Coordination between the Unfolded Protein Response and ER-Associated Degradation , 2000, Cell.

[49]  Sangram S. Sisodia,et al.  Dual Roles for Ste24p in Yeast a-Factor Maturation: NH2-terminal Proteolysis and COOH-terminal CAAX Processing , 1998, The Journal of cell biology.

[50]  S. Prusiner,et al.  A transmembrane form of the prion protein in neurodegenerative disease. , 1998, Science.

[51]  P. Ross-Macdonald,et al.  Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. , 1994, Genes & development.

[52]  D. Botstein,et al.  A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. , 1987, Gene.

[53]  D. Garfinkel,et al.  Single-step selection for Ty1 element retrotransposition. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[54]  Kei-Hoi Cheung,et al.  Large-scale analysis of the yeast genome by transposon tagging and gene disruption , 1999, Nature.