Date of Degree

1999

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Corinne A. Michels

Committee Members

Harold Magazine

Timothy Short

Dan Eshel

Lorraine Marsh

Subject Categories

Biology

Abstract

The addition of glucose to maltose fermenting Saccharomyces cerevisiae cells results in the rapid loss of maltose transport activity. This results both from the repression of the maltose permease gene transcription and from the post-translational inactivation of maltose permease through a process termed glucose-induced inactivation of maltose permease. The inactivation consists of two separable processes, a rapid inhibition of maltose transport activity and by a slower degradation of maltose permease protein. Degradation is dependent on endocytosis, vesicular sorting and vacuolar proteolysis, and is independent of the proteasome. Furthermore, maltose permease exists in differentially phosphorylated forms.

Ubiquitin and the ubiquitin-conjugating system are necessary for glucose-induced proteolysis of maltose permease. The rate of glucose-induced proteolysis of maltose permease is slowed by deletion of DOA4 encoding a ubiquitin recycling enzyme and this is suppressed by overexpression of ubiquitin. Moreover, maltose permease exists in a ubiquitinated state whose abundance increases upon the addition of glucose. A mutation that reduces the expression of NPI1/RSP5, encoding a putative ubiquitin-protein ligase, also dramatically decreases the rate of glucose-induced proteolysis of maltose permease. Double null mutations in ubc1 ubc4 and ubc4 ubc5 had no significant impact on the rate of glucose-induced proteolysis of maltose permease.

Mutational analysis was carried out on the predicted cytoplasmic N- and C-terminal domains of maltose permease. The N-terminal cytoplasmic domain of maltose permease contains a putative PEST sequence and a dileucine sequence. The C-terminal cytoplasmic domain contains a putative endocytosis signal, NPF, and numerous lysine, serine, and threonine residues. The PEST sequence is required for rapid glucose-induced inhibition and degradation of maltose permease while alterations in the C-terminal domain cause only a modest decrease.

In summary, our results indicate that glucose stimulates the ubiquitination of maltose permease and that this marks this protein for endocytosis and vacuolar proteolysis. The PEST sequence found in residues 49–78 of the N-terminal cytoplasmic domain plays an essential role in this process and appears to be required for recognition by the ubiquitin conjugating enzymes.

Comments

Digital reproduction from the UMI microform.

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Biology Commons

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