Date of Award

5-2006

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Jeffrey M. Becker

Committee Members

David A. Brian, Pamela L. C. Small, Todd Reynolds, Bruce McKee

Abstract

Dipeptides and tripeptides serve as important sources of amino acids, nitrogen and carbon for the growth of all organisms. To identify genes involved in the regulation of small peptide utilization, I performed a systematic, functional examination of this process in a haploid, non-essential, single-gene deletion mutant library of Saccharomyces cerevisiae. In addition, we used high-throughput phenotyping in which we grew yeast cells on 284 different dipeptides or 11 tripeptides as the sole nitrogen source to dissect different mechanisms of di/tri-peptide utilization in seven genetically diverse strains. I have identified 103 candidate genes involved in regulating peptide utilization: 57 genes whose deletion decreased dipeptide utilization and 46 genes whose deletion enhanced dipeptide utilization. Since in S. cerevisiae the membrane transport protein Ptr2p, encoded by PTR2, mediates the uptake of di/tri-peptides and is a key player in the di/tri- peptide utilization process, I focused our studies on the regulation of PTR2 expression, Ptr2p-GFP localization, and dipeptide uptake assays. Forty-two genes were ascribed to the regulation of PTR2 expression, 37 genes were involved in Ptr2p localization, and 24 genes apparently did not affect expression or localization. Together with the high- throughput phenotyping study, we have identified gene components involved in regulation of dipeptide utilization in many cellular processes: 1) proteins involved in up- or down-regulation of PTR2 transcription including the chromatin remodeling INO80 protein complex (Arp5p, Arp8p, Ies6p and Taf14p), transcription factors (Cup9p, Rpn4p, Stp2p, and Dal81p), polymerase mediators (Ssn3p/Srb10p, Ssn8p/Srb11p, Ssn2p/Srb9p, and Srb8p), and mRNA maturation (Kem1p and Pat1p); 2) proteins involved in the Ptr2p trafficking system including ESCRT I, II, III protein complex; 3) proteins participating in metabolic processes including the pyruvate dehydrogenase complex (Pdx1p, Lpd1, Pdb1p, and Lat1p), the fatty acid synthase complex (Oar1p, Mct1p, and Etr1p), and the glycine decarboxylase multienzyme complex (Gcv2p, Gcv3p, and Lpd1p); 4) proteins involved in other cellular processes, such as Ybt1p, a protein located at the vacuole membrane likely related to the storage of dipeptides in the cytosol and Dal5p previously identified as an allantoate/ureidosuccinate permease, which facilitates di/tri-peptide transport. Specifically, Dal5p is involved in the uptake of non-N-end rule dipeptides. Moreover, even in the absence of Dal5p and Ptr2p, an additional activity - almost certainly the periplasmic asparaginase II Asp3p - facilitates the utilization of dipeptides with C-terminal asparagine residues by a different strategy. Another, as yet unidentified activity, enables the utilization of dipeptides with C-terminal arginine residues. The identified genes regulating dipeptide utilization were distributed among most of the Gene Ontology functional categories indicating a very wide regulatory network involved in transport and utilization of dipeptides in yeast. It is anticipated that further characterization of how these genes affect peptide utilization should add new insights into the global mechanisms of regulation of transport systems in general and peptide utilization in particular.

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