Doctoral Dissertations

Winter 1995

Dissertation

Biochemistry

Degree Name

Doctor of Philosophy

Clyde L Denis

Abstract

In yeast, Sacchoromyces cerevisiae, transcription of $\underline{ADH2}$ gene (encodes alcohol dehydrogenase II) under glucose derepression conditions requires not only the gene-specific activator, Adr1p, but also the general transcriptional factors like Ccr4p. Ccr4p belongs to the LRR-containing protein superfamily, members of which have often been found to associate with other proteins to form a functional protein complex. This dissertation reports three major conclusions from my research.

1. The CCR4 protein is associated with at least three other proteins by immunoprecipitation using the CCR4 antibody, and more importantly, the LRR in the CCR4 protein is not only essential, but also sufficient to form the CCR4 complex.

2. Using the yeast two hybrid procedure with LexA-CCR4 as a bait, seven genes were isolated. Two of them are known genes, $\underline{\rm CAF1}$ and $\underline{\rm DBF2},$ respectively. Three of them encoding novel proteins contain the known structural motifs: ATP-binding motif for $\underline{\rm CAF16},$ zinc-finger DNA-binding motif for $\underline{\rm CAF10},$ and WD40 repeats for $\underline{\rm CAF4.\ CAF6}$ and $\underline{\rm CAF17}$ showed no sequence similarity to any genes in the current Data Base. Using LexA-CAF16 as a bait to perform the yeast two hybrid procedure, four genes, encoding Caf16p, Map1p, Srb9p and Mth1p were also identified.

3. The CCR4 complex is purified by using 6His tagged $\underline{\rm CCR4}$ and $\underline{\rm CAF1}.$ The purified CCR4 complex from the CAF1-6His containing whole cell extract through three chromatographic procedures has a molecular weight of 1.0 $\times$ 10$\sp6$ Da, including Ccr4p, Caf1p, Caf16p, Caf17p, Dbf2p and other unidentified proteins such as 185 kDa, 145 kDa and 110 kDa proteins.

These results give us an inside view into understanding how CCR4 complex is involved in diverse cellular processes, and strongly suggests that CCR4 complex affects different cellular events by interacting with other functional proteins or protein complexes.

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