Mark D. Rose
The yeast Saccharomyces cerevisiae is an ideal organism for the combined genetic and biochemical analysis of fundamental cell processes universal to eukaryotic cells. The major goals of our research are the dissection of the mechanisms of cell and nuclear fusion during mating and the pathway of spindle pole body duplication. During mating, yeast cells respond to gradients of mating pheromone by growing toward one another in a polarized fashion. After adhering together, they remove their cell walls in the region of close contact and fuse their plasma membranes to produce a single cell. As cell fusion progresses, the two nuclei become connected by microtubules emanating from the organizing centers (spindle pole bodies). The nuclei then move together in a microtubule-motor-driven process. Subsequently, nuclear envelopes fuse in the vicinity of the spindle pole bodies, producing a single diploid nucleus. Accordingly, cell and nuclear fusion serve as powerful indicators of a variety of basic cell biological processes, including cell polarization, membrane fusion, nuclear movement, and microtubule organization. Using novel genetic screens, we have identified several genes required for cell and nuclear fusion. Using modern molecular genetic techniques, we are identifying and characterizing the proteins and their biological functions. A detailed analysis of the pathway of cell and nuclear fusion will be important for the understanding of a broad set of phenomena common to all eukaryotic cells. Descriptions of representative genes follow.
KAR9 is required for the orientation of cytoplasmic microtubules in both mating and mitosis. Kar9p acts as a protein "adapter" that tethers the cytoplasmic microtubules to a cortical actin-associated site in the yeast bud. In mitosis, the motor protein dynein then pulls the nucleus into the bud. In mating cells, Kar3p pulls the nucleus into the region of cell fusion.
KAR1 encodes an essential component of the spindle pole body (SPB) required both for nuclear fusion and for SPB duplication and different protein domains mediate these two functions. One domain of Kar1p interacts with Cdc31p, an essential and highly conserved centrosomal protein also called centrin. Centrins are ubiquitous in eukaryotic microtubule organizing centers including those of plants, fungi and humans. Surprisingly, we found that Cdc31p also functions in cell morphogenesis via interactions with a conserved kinase called Kic1p. Recent work has utilized fine structural genetic analysis to identify residues of Cdc31p required for its different functions.
KAR3 encodes a minus-end oriented microtubule-dependent motor protein related to kinesin. Kar3p and its associated light chain Cik1p form the motor responsible for moving nuclei together in zygotes. Kar3p also plays a critical role in mitosis, where it counteracts the activities of other kinesin-related proteins. A novel transcriptional regulator, Kar4p, is required for the specific induction of Kar3p and Cik1p during mating. Current work is directed at dissecting the network of Kar4p regulated genes.
Ultimately, the two SPBs come into close apposition and the nuclear envelopes fuse in a process called homotypic membrane fusion. Several proteins resident in the nuclear envelope, including Kar2p, Kar5p, Kar7p, and Kar8p, are required for nuclear membrane envelope fusion. Of these, only Kar5p is specifically induced during mating and then localizes to the site of membrane fusion. The specific role of Kar5p in nuclear envelope fusion is under active investigation.
Kel1p Mediates Yeast Cell Fusion Through a Fus2p- and Cdc42p-Dependent Mechanism. Genetics. 2016 ;202(4):1421-35. .
An Amphiphysin-Like Domain in Fus2p Is Required for Rvs161p Interaction and Cortical Localization. G3 (Bethesda). 2015 ;6(2):337-49. .
Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling. PLoS Genet. 2015 ;11(12):e1005684. .
ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative, Sey1p-independent homotypic ER fusion pathway. Mol Biol Cell. 2014 ;25(21):3401-12. .
Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae. G3 (Bethesda). 2014 ;5(1):111-21. .