Research Interests

The lab exploits genetic, molecular and biochemical approaches using the budding yeast Saccharomyces cerevisiae to address issues of eukaryotic cell regulation and control of cell proliferation. The yeast is a particularly suitable organism for studies of basic cell function since yeast can be manipulated easily at the molecular and genetic levels, and yeast has been shown repeatedly to display a high degree of functional conservation for a wide variety of cellular functions including cell-cycle regulation, metabolism, gene expression and morphogenesis. Thus, findings with this genetically tractable organism have provided (and will undoubtedly continue to provide) insight into the performance and regulation of a spectrum of cellular activities in all cells including mammalian cells.

Our general approach to issues such as control of cell proliferation has exploited yeast genetics to first identify mutations that affect aspects of proliferation. We then explore these mutational situations using molecular and biochemical strategies. Over the past few years this fruitful genetic strategy has led us to focus on two major issues.

Global gene expression in the context of chromatin

As the result of a genetic search for mutations that affect cell proliferation, we identified the cdc68-1 mutation that imposes a conditional (temperature-sensitive) phenotype on mutant cells: proliferating cdc68-1 mutant cells shifted to a high temperature are able to complete all aspects of an ongoing cell cycle but are not able to complete the cell-cycle regulatory step termed START. Mutant cells therefore become uniformly arrested at START. Completion of START involves activation of a highly conserved protein kinase that is regulated by association with regulatory proteins termed cyclins. We found that Cdc68 is required for expression of cyclin genes so that impaired Cdc68 function precludes activation of this kinase and results in arrest of proliferation. We now know that Cdc68 both activates and represses expression of a wide spectrum of genes, and probably affects gene expression through remodeling chromatin structure.

We have found that the Cdc68 protein is a component of an abundant nuclear complex, and is found in association with another protein, Pob3. The highly conserved Cdc68/Pob3 complex (the CP complex) is as abundant as the number of nucleosomes. The analogous mammalian complex remodels chromatin for transcription in vitro, and our in vivo genetic analysis shows that this CP complex both activates transcription and mediates the repression of transcription that is conferred by chromatin. We are continuing to explore the function of Cdc68 using both genetic and biochemical approaches to define other proteins that affect Cdc68 activity, and to determine the protein-protein interactions that are required for CP complex function.

Vesicular transport and the resumption of cell proliferation

When limiting nutrients, yeast cells cease proliferation in a regulated manner, and such non-proliferating cells are referred to as stationary-phase cells. We identified a conditional (cold-sensitive) mutation, termed gcs1-1, that causes stationary-phase cells to become defective for resumption of proliferation upon addition of fresh medium at the restrictive temperature of 15°C. If such gcs1 mutant cells are first allowed to resume proliferation at a permissive temperature (30°C) and then transferred to 15°C, mutant cells now continue to proliferate so long as nutrients are sufficient. Thus the Gcs1 protein is involved in the transition from stationary phase to active proliferation and is not required to maintain proliferation once initiated. To investigate this interesting activity, we have cloned the GCS1 gene and studied its function.

In collaboration with several other labs, we have found that Gcs1 is a component of the highly conserved vesicle transport system that provides directed and regulated movement of vesicles and cargo among cellular compartments. Formation of vesicles and subsequent docking and fusion of vesicles with appropriate target membranes involves a class of GTP-binding protein termed ARF. Unlike many G-proteins, ARF proteins do not possess any intrinsic GTPase activity to modulate their function, and thus must rely on a GTPase-activating protein (GAP) to hydrolyse GTP and so allow vesicle fusion. We have shown that the Gcs1 protein is a GAP for several yeast ARF proteins, and defines a structurally and functionally conserved class of Zn-finger proteins.

Another protein, Glo3, with structural similarity to Gcs1 also has ARF GAP activity in vitro and functions in an overlapping fashion with Gcs1 to facilitate retrograde transport from the Golgi to the ER. The Gcs1 protein functions at other vesicular transport stages in addition to the Golgi - ER shuttle, and we are investigating the additional sites of Gcs1 function by characterizing other proteins with structural similarities to Gcs1 and Glo3, and we are using genetic and molecular strategies to identify other proteins involved in ARF GAP function. In addition to gaining a greater appreciation of vesicular transport in eukaryotic cells, we will also be able to explore the role of vesicular transport in the transition from stationary phase (quiescence) to active cell proliferation.