Characterisation of a negative regulator of hydrophobic amino acid transport in Saccharomyces cerevisiae.
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Yeast have many systems for nitrogen regulation. Combined, these systems make a complex network balancing uptake of nitrogenous compounds and their assimilation in diverse environments. Yeast preferentially use more easily assimilated nitrogen sources when they are available, and under these conditions, many permeases that transport less easily utilisable amino acids are inactive. This study characterises a spontaneous mutation that results in deregulation of an apparently unique hydrophobic amino acid permease (entitled Lup for Leucine uptake), in the presence of ammonium. The mutant, Lup+ phenotype (ability to grow in leucine limiting conditions), was recessive and postulated to be due to a mutation in a gene, designated LUP1, which encodes a represser of the Lup permease. Since Lup+ cells better accumulated hydrophobic amino acids, we used toxic amino acid analogs in growth media to select for LUP1 (or LUP/lup1) genotypes. Interestingly, we have observed that Lup+ variants were more sensitive than their progenitor to not only the expected hydrophobic amino acid analogs L-methionine sulfoximine, L-ethionine and m-fluoro-D,L-phenylalanine, but also the nonhydrophobic analogs L-canavanine and L-azaserine. A screen of two wildtype yeast genomic libraries has identified 14 plasmids that complement the m-fluoro-D,Lphenylalanine sensitive (Fpas) phenotype of the lup1 allele. Sequence data of complementing plasmids, extending outwards from transposons whose insertions defined the physical size of the complementing unit, has revealed that two genes, BUL1 and AR04, can complement the Fpas phenotype. BUL1, but not AR04, could also render Lup+ cells Lup- (unable to grow in limited leucine environments), thus was predicted to be allelic to LUP1. Several additional lines of evidence demonstrated that LUP1 and BUL1 were allelic: (i) partial deletion of chromosomally encoded BUL1resulted in Lup+ phenotypes; (ii) like bull mutants, lup1 mutants were temperature sensitive; (iii) LUP1 and BUL1 were in the same, or extremely close, chromosomal position; and (iv) Lup+ mutants had an altered BUL1 sequence to wildtype. The nature of changes to the BUL1 sequence occurring in two Lup+ variants consisted of point mutations occurring at different positions. Bull is thought to be involved in the ubiquitination pathway due to physical interaction with the Rsp5 ubiquitin ligase. Rsp5 has been implicated directly with the ubiquitin-dependent internalization and down-regulation of at least four yeast plasma membrane proteins. Rsp5 may also be involved in regulation of the Lup permease as a mutation in Lup1/Bul1 that eliminates its ability to bind to Rsp5 also abolishes its capacity to complement the Lup+ and Fpas phenotypes. Based on these phenotypes, the following model was proposed: Lup1/Bul1 functions with Rsp5 as an E3 complex for the recognition and subsequent ubiquitination of the Lup permease, targeting this protein for destruction in the presence of ammonium.