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Vallier, L. G., D. Coons, L. F. Bisson, & M. Carlson (1994) Altered regulatory responses to glucose are associated with a glucose transport defect in grrl mutants of Saccharomyces cerevisiae. Genetics 136: 1279-1285.

Article 11 introduces a new player in the glucose sensing/signal transduction pathway called GRR1 for glucose repression resistant (see Article 11, References section, for references), grrl mutations are pleiotropic. Strains carrying grrl mutations exhibit a number of phenotypic defects. These include glucose-insensitive transcription of the normally glucose repressed GAL, MAL, and SUC2 genes; an extremely elongated shape rather than the normal ovoid morphology; slow growth on glucose; increased sensitivity to osmotic stress and nitrogen starvation; decreased divalent cation transport; and growth defects in aromatic amino acid auxotrophs. These pleiotropic defects suggest quite strongly that the Grrl protein plays a regulatory role in many cellular pathways.

The Grrl protein is quite large (132 kDaltons) and contains 12, 26-residue leucine-rich repeats. X-ray crystallographic analysis of other leucine-rich repeat proteins demonstrates that the overall structure is that of a thick horseshoe with each of the 12 repeats forming a beta-sheet and these arranged so that they are exposed on the outer surface of the horseshoe curve. Grrlp and other proteins in this class are known to interact with several other proteins by means of these repeats to form large multiprotein complexes. Recent evidence (Li & Johnston, 1997) shows that Grrlp is part of a large protein complex with ubiquitin-protein ligase-like activity and is involved in the degradation of certain proteins, including cell cycle regulators.

1. The results in Figure 1 demonstrate that grrl A mutant strains are deficient in high-affinity glucose transport, rgtl-1 partially restores the level of high-affinity glucose transport in the grrl A mutant strain. The results in Figure 2 are consistent with this finding.

(a) What phenotype is being observed in Figure 2?

(b) Why are these results consistent with those in Figure 1 regarding the grrl A strain and the grrl A rgtl-1 double mutant strain?

(c) Figure 2 shows that the snf3 A mutation enhances the grrl A phenotype. What result demonstrates this?

(d) Is rgtl-1 epistatic to grrl A'! To snf3Al What data support your conclusion?

2. Specifically, what result reported in Table 3 indicates that rgtl-1 restores the glucose repression sensitivity of grrl A. mutants?

3. Does rgtl-1 suppress all grrl phenotypes?

4. Briefly, why do the authors say that grrl mutations do not by-pass regulation by SNF1, SNF2, SNF4, and SNF5?

5. Specifically, what evidence is presented in Table 4 to indicate that ssn6A mutations suppress the glucose-repression resistance of invertase expression in grrl A mutation strains?

REFERENCE

Li, F. & M. Johnston (1997) Grrlof Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skpl: coupling glucose sensing to gene expression and the cell cycle. EMBO J. 16: 5629-5638.

CASE STUDY I ARTICLE 12

Ozcan, S., T. Leong, & M. Johnston (1996) Rgtlp of Saccharomyces cerevisiae, a key regulator of glucose-induced genes, is both an activator and a repressor of transcription. Mol. Cell. Biol. 16: 6419-6426.

1. Describe the method used to clone RGT1. Be sure to include the genotype of the host strain, a brief description of the library (vector and genotype of strain providing the insert fragments), the structure of the reporters, and the pheno-type used to identify transformant clones containing RGT1.

2. How did the authors use targeted integration to demonstrate that they had cloned the real RGT1 gene?

3. What interesting structural feature of Rgtlp was revealed by its sequence?

4. According to their previous publications, Rgtlp repressed expression of several HXT genes encoding glucose transporters. How did they demonstrate that Rgtlp repression of HXT1 is direct, i.e. results from the binding of Rgtl protein to the HXT1 promoter?

5. Figure 3, Panel A, lines 1 and 2 demonstrate that Rgtlp is both a repressor (in the absence of glucose) and an activator (in 4% glucose). Discuss these data.

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