The Saccharomyces cerevisiae mating pheromone a-factor is an extracellular signaling molecule whose biogenesis pathway is distinctive. The a-factor precursor undergoes post-translational addition of an isoprene lipid (farnesyl), methylation, and several proteolytic processing steps to produce a prenyl- and methyl-modified oligopeptide. a-Factor does not exit cells via the classical secretory pathway which involves intracellular secretory organelles, but instead utilizes a novel membrane transport pump for export. Several components of the a-factor biogenesis machinery are known: the RAM1 and RAM2 gene products together comprise the farnesyltransferase which mediates a-factor prenylation, STE14 encodes the methyltransferase, and STE6 encodes the a-factor export pump. The overall goal of this proposal is to gain a high resolution view of a-factor biogenesis by investigating modification, processing, and secretion of a-factor in detail and examining how these events are co-ordinated. Molecular, genetic, and biochemical approaches will be used to probe the activity of known biogenesis components, genetically identify new components, and examine protein-protein interactions between a-factor and its biogenesis machinery.
Specific aims are: 1) to identify recognition determinants in a-factor by mutational analysis; 2) to examine the extent to which farnesyl is a specific biogenesis determinant by testing whether related lipids can substitute for it in a-factor; 3) to ascertain the cellular location of the STE14 methyltransferase, and further examine its role in a-factor biogenesis; 4) to genetically identify the genes involved in N-terminal processing of a-factor; 5) to determine residues within the STE6 transporter that are critical for a-factor recognition; 6) to characterize a novel a-factor-related peptide (AFRP) that exits cells by a STE6-independent pathway; and 7) to use heterologous systems to examine the biogenesis of a-factor and related pheromones produced by other yeast species. The study of a-factor will provide insight into a variety of cellular mechanisms that are medically relevant including prenylation and methylation of Ras oncoproteins, efflux of chemotherapeutic drugs by the mammalian multidrug resistance protein ( a close relative of the a-factor transporter STE6), and processing and membrane traversal of peptides involves in antigen presentation by the immune system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041223-06
Application #
2180725
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1988-12-01
Project End
1996-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
6
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Ast, Tslil; Michaelis, Susan; Schuldiner, Maya (2016) The Protease Ste24 Clears Clogged Translocons. Cell 164:103-114
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Michaelis, Susan; Hrycyna, Christine A (2013) Biochemistry. A protease for the ages. Science 339:1529-30
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Michaelis, Susan; Barrowman, Jemima (2012) Biogenesis of the Saccharomyces cerevisiae pheromone a-factor, from yeast mating to human disease. Microbiol Mol Biol Rev 76:626-51
Barrowman, Jemima; Hamblet, Corinne; Kane, Megan S et al. (2012) Requirements for efficient proteolytic cleavage of prelamin A by ZMPSTE24. PLoS One 7:e32120
Barrowman, Jemima; Bhandari, Deepali; Reinisch, Karin et al. (2010) TRAPP complexes in membrane traffic: convergence through a common Rab. Nat Rev Mol Cell Biol 11:759-63

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