Molecular Biology Faculty
Mark D. Rose
Lewis Thomas Lab, 319
Lab (609) 258-2805
Cell biology and genetics in Saccharomyces cerevisiae
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.
Rogers JV, Arlow T, Inkellis ER, Koo TS, Rose MD. (2013) ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating. Mol Biol Cell. Oct 23. [Epub ahead of print]
Kim J, Rose MD. (2012) A mechanism for the coordination of proliferation and differentiation by spatial regulation of Fus2p in budding yeast. Genes Dev. 26: 1110-1121. PubMed
Ydenberg CA, Stein RA, Rose MD. (2012) Cdc42p and Fus2p act together late in yeast cell fusion. Mol Biol Cell. 23: 1208-1218. PubMed
Melloy P, Shen S, White E, Rose MD. (2009) Distinct roles for key Karyogamy proteins during yeast nuclear fusion. Mol Biol Cell. 20: 3773-3782. PubMed
Sheltzer JM, Rose MD. (2009) The class V myosin Myo2p is required for Fus2p transport and actin polarization during the yeast mating response. Mol Biol Cell. 20: 2909-2919. PubMed
Shen S, Tobery CE, Rose MD. (2009) Prm3p Is a Pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion. Mol Biol Cell. 20: 2438-2450. PubMed
Hayes AP, Sevi LA, Feldt MC, Rose MD, Gammie AE. (2009) Reciprocal regulation of nuclear import of the yeast MutSalpha DNA mismatch repair proteins Msh2 and Msh6. DNA Repair (Amst). 8: 739-751. PubMed
Ydenberg CA, Rose MD. (2009) Antagonistic regulation of Fus2p nuclear localization by pheromone signaling and the cell cycle. J Cell Biol. 184: 409-422. PubMed
Ydenberg CA, Rose MD. (2008) Yeast mating: a model system for studying cell and nuclear fusion. Methods Mol Biol. 475: 3-20. PubMed
Paterson JM, Ydenberg CA, Rose MD. (2008) Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating. J Cell Biol. 181: 697-709. PubMed
Melloy P, Shen S, White E, McIntosh JR, Rose MD. (2007) Nuclear fusion during yeast mating occurs by a three-step pathway. J Cell Biol 179: 659-670. PubMed
Gammie AE, Erdeniz N, Beaver J, Devlin B, Nanji A, Rose MD. (2007) Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae. Genetics 177: 707-721. PubMed
Lahav R, Gammie A, Tavazoie S, Rose MD. (2007) Role of transcription factor Kar4 in regulating downstream events in the Saccharomyces cerevisiae pheromone response pathway. Mol Cell Biol 27: 818-829. PubMed
Clark SW, Rose MD (2005). Arp10p is a pointed-end-associated component of yeast dynactin. Mol Biol Cell. 17: 738-748. PubMed
Clark SW, Rose MD (2005). Alanine scanning of Arp1 delineates a putative binding site for Jnm1/dynamitin and Nip100/p150Glued. Mol Biol Cell 16: 3999-4012. PubMed
Fitch PG, Gammie AE, Lee DJ, de Candal VB, Rose MD (2004). Lrg1p Is a Rho1 GTPase-activating protein required for efficient cell fusion in yeast. Genetics 168: 733-746. PubMed
Matheos D, Metodiev M, Muller E, Stone D, Rose MD (2004). Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p. J Cell Biol 165: 99-109. PubMed
Kabani M, Kelley SS, Morrow MW, Montgomery DL, Sivendran R, Rose MD, Gierasch LM, Brodsky JL (2003). Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP. Mol Biol Cell 4: 3437-3448. PubMed
Kimata Y, Kimata YI, Shimizu Y, Abe H, Farcasanu IC, Takeuchi M, Rose MD, Kohno K (2003). Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins. Mol Biol Cell 14: 2559-2569. PubMed
Rose M (2003). The awesome power of genetics education. Cell Biol Educ 2: 96-97. PubMed
Metodiev MM, Matheos D, Rose MD and Stone DE (2002). Regulation of the yeast pheromone response by direct interaction between the mating-specific Ga and MAPK proteins. Science 296: 1483-1486. PubMed
Gammie AE and Rose MD (2002). Assays of cell and nuclear fusion. Meth Enzymol 351: 477-498. PubMed
Ivanovska I and Rose MD (2001). Fine structure analysis of the yeast centrin, Cdc31p, identifies residues specific for cell morphology and spindle pole body duplication. Genetics 157: 503-518. PubMed
Khalfan W, Ivanovska I and Rose MD (2000) Functional interaction between the PKC1 pathway and CDC31 network of SPB duplication genes. Genetics 155: 1543-1559. PubMed
Miller RK, Cheng SC, Rose MD (2000). Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules. Mol Biol Cell 11: 2949-2959. PubMed
Bishop AC, Ubersax JA, Petsch DT, Matheos DP, Gray NS, Blethrow J, Shimizu E, Tsien JZ, Schultz PG, Rose MD, Wood JL, Morgan DO, Shokat KM (2000). A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407: 395-401. PubMed
Brizzio V, Khalfan W, Huddler D, Beh CT, Andersen SS, Latterich M, Rose MD (1999). Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5 and KAR2 during nuclear fusion in Saccharomyces cerevisiae. Mol Biol Cell 10: 609-626. PubMed
Miller RK, Matheos D, Rose MD (1999). The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization. J Cell Biol 144: 963-975. PubMed
Gammie AE, Stewart BG, Scott CF, Rose MD (1999). The two forms of the karyogamy transcription factor, Kar4p, are regulated by differential initiation of transcription, translation and protein turnover. Mol Cell Biol 19: 817-825. PubMed