真菌類的侵入性生長會影響其致病能力，其調控機制在過去已被研究的相當廣泛。Bud2p可以幫助Bud1p/Rsr1p之鳥糞嘌呤核苷三磷酸水解能力，Bud2p在過去的研究中指出會參與在酵母菌出芽生殖及侵入性生長的過程中，但是詳細的分子機制目前還尚未了解。本研究指出：在酵母菌侵入性生長的過程中Arf3p可以調控Bud2p的活性。我們發現Arf3p藉由直接作用於Bud2p的胺基端來促進其酵素活性，透過遺傳學及生化分析也發現Bud1p的活化態及不活化態可以影響酵母菌侵入性生長，且當細胞中缺乏Arf3p或是Bud2p都會讓細胞中累積過多的活化態Bud1p。此外，我們也發現當葡萄糖缺乏所引發的酵母菌侵入性生長中，會增加活化態Arf3p，且此過程與其鳥糞嘌呤核苷酸交換因子Yel1p無關。因此我們提出一個機制，那就是當酵母菌遭遇到葡萄糖缺乏時，其會促進Arf3p活化，並會與Bud2p直接作用來調控Bud2p對於提升Bud1p之鳥糞嘌呤核苷三磷酸水解能力，進而影響酵母菌的侵入性生長。

The regulation and signaling pathways involved in the invasive growth of yeast have been studied extensively because of their general applicability to fungal pathogenesis. Bud2p, which functions as a GTPase-activating protein (GAP) for Bud1p/Rsr1p, is required for appropriate budding patterns and filamentous growth. However, the regulatory mechanisms leading to Bud2p activation are poorly understood. In this study, we report that Arf3p acts as a regulator of Bud2p activation during invasive growth. Arf3p binds directly to the N-terminal region of Bud2p and promotes its GAP activity both in vitro and in vivo. Genetic analysis shows that deletion of BUD1 suppresses the defect of invasive growth in arf3 or bud2 cells. Lack of Arf3p, like that of Bud2p, causes the intracellular accumulation of Bud1p-GTP. The Arf3p-Bud2p interaction is important for invasive growth and facilitates the Bud2p-Bud1p association in vivo. Finally, we show that under glucose depletion-induced invasion conditions in yeast, more Arf3p is activated to the GTP-bound state and the activation is independent of Arf3p guanine nucleotide-exchange factor (GEF) Yel1p. Thus, we demonstrate that a novel spatial activation of Arf3p plays a role in regulating Bud2p activation during glucose-depletion-induced invasive growth.

摘要……………………………………………………………………………………4
Abstract………………………………………………………………………………5
Abbreviations…………………………………………………………………………6
Chapter I. Literature Review…………………………………………………………7
1. Small GTP-binding Proteins…………………………………………………7
2. ADP-ribosylation Factors……………………………………………………10
3. Cell Polarity…………………………………………………………………16
Chapter II. Arf3p GTPase is a key regulator of Bud2p activation for haploid yeast invasive growth………………………………………………………………………26
1. Introduction…………………………………………………………………27
2. Results………………………………………………………………………28
3. Discussion……………………………………………………………………37
4. Materials and Methods………………………………………………………40
Tables…………………………………………………………………………………45
Table 1. Yeast strains used in this study…………………………………………45
Table 2. Plasmids used in this study…………………………………………46
Figures………………………………………………………………………………48
Figure 1. Arf3p is required for yeast invasive growth…………………………48
Figure 2. Arf3p specifically and directly interacts with Bud2p………………49
Figure 3. Arf3p associates with Bud2p in vivo…………………………………50
Figure 4. Schematic representation of the various constructs containing the BUD2 truncation mutants……………………………………………51
Figure 5. Arf3p interacts directly with the N-terminal region of Bud2p in a GTP-dependent manner………………………………………………52
Figure 6. Arf3p-GTP specifically interacts with the N-terminus of Bud2p in vitro…………………………………………………………………53
Figure 7. Bud2p interacts with Arf3pQ71L via the N-terminal region…………54
Figure 8. Bud2p-dN40 dissociates from Arf3p in vivo…………………………55
Figure 9. The N-terminus of Arf3p interacts with Bud2p………………………56
Figure 10. Alignment of Arf3p with other ARF protein family members………57
Figure 11. Polarization to daughter cell membrane and heavy membrane enrichment of Arf3p-GFP, Arf3pL23V-GFP, and Arf3pI33V-GFP……58
Figure 12. Isoleucine 33 of Arf3p is essential for the interaction of Arf3p with Bud2p………………………………………………………………59
Figure 13. Both Arf3pL23V and Arf3pI33V interact with Ye1lp and Afi1p………60
Figure 14. Arf3p and Bud2p independently localize to the bud neck…………61
Figure 15. Arf3p interacts with Bud2p to regulate yeast invasive growth……62
Figure 16. Bud2p-dN40 localizes to the bud neck……………………………63
Figure 17. The budding pattern seen with Bud2p-dN40 expression in bud2
Figure 18. Arf3p interacts with Bud2p to regulate yeast invasive growth……65
Figure 19. Yeast invasion is not altered by disruption of BUD1 or BUD5……66
Figure 20. Overexpression of active forms of Bud1p results in loss of agar invasion……………………………………………………………67
Figure 21. Depletion of BUD1 suppresses the invasive growth defect of arf3 and bud2 cells……………………………………………………68
Figure 22. Accumulation of Bud1p-GTP prevents yeast invasive growth……69
Figure 23. L681A and R682Q mutations in Bud2p (Bud2pAQ) cause defects in GAP activity………………………………………………………70
Figure 24. Bud2p GAP activity, which stimulates Bud1p-GTP hydrolysis, is required for both polarity establishment and yeast invasive growth………………………………………………………………71
Figure 25. GST-Cdc42-GDP specifically pulls down Bud1pG12V………………72
Figure 26. Elevated Bud1p-GTP levels were detected in arf3 and bud2 mutant cells…………………………………………………………………73
Figure 27. Bud2p interacts with Arf3p to enhance GAP activity leading to Bud1p-GTP hydrolysis in vivo……………………………………74
Figure 28. Interaction of Arf3p and Bud2p promotes Bud2p GAP activity leading to Bud1p GTP hydrolysis…………………………………………75
Figure 29. Arf3p facilitates the Bud2p-Bud1p association in vivo……………76
Figure 30. Arf3p is required for yeast invasive growth in response to glucose depletion……………………………………………………………77
Figure 31. Arf3p is activated by glucose depletion……………………………78
Figure 32. Arf3p and Bud2p independently polarize to the bud tip in response to glucose depletion……………………………………………………79
Figure 33. Glucose depletion induces Arf3p activation to promote Bud1p-GTP hydrolysis…………………………………………………………80
Figure 34. Yel1p is not required for agar invasion……………………………81
Figure 35. Arf3p associates with Bud2p in yel1 cells…………………………82
Figure 36. Disruption of YEL1 in yeast cells shows signals that polarize Arf3p-GFP to the bud tip in the absence of glucose………………83
Figure 37. Active forms of Arf3p are elevated in yel1 cells upon glucose depletion……………………………………………………………84
Figure 38. Gea1p, Gea2p, and Syt1p are not required for yeast invasive growth………………………………………………………………85
Figure 39. A model for the roles of Arf3p in budding polarity and yeast invasion……………………………………………………………86
References……………………………………………………………………………87

Adam, S.A. (1999). Transport pathways of macromolecules between the nucleus and the cytoplasm. Curr Opin Cell Biol 11, 402-406.

Adames, N., Blundell, K., Ashby, M.N., and Boone, C. (1995). Role of yeast insulin-degrading enzyme homologs in propheromone processing and bud site selection. Science 270, 464-467.

Anthonio, E.A., Brees, C., Baumgart-Vogt, E., Hongu, T., Huybrechts, S.J., Van Dijck, P., Mannaerts, G.P., Kanaho, Y., Van Veldhoven, P.P., and Fransen, M. (2009). Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6. BMC Cell Biol 10, 58.

Baschieri, F., and Farhan, H. (2012). Crosstalk of small GTPases at the Golgi apparatus. Small GTPases 3, 80-90.

Bauer, Y., Knechtle, P., Wendland, J., Helfer, H., and Philippsen, P. (2004). A Ras-like GTPase is involved in hyphal growth guidance in the filamentous fungus Ashbya gossypii. Mol Biol Cell 15, 4622-4632.

Bender, A. (1993). Genetic evidence for the roles of the bud-site-selection genes BUD5 and BUD2 in control of the Rsr1p (Bud1p) GTPase in yeast. Proc Natl Acad Sci U S A 90, 9926-9929.

Bender, A., and Pringle, J.R. (1989). Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci U S A 86, 9976-9980.

Booden, M.A., Baker, T.L., Solski, P.A., Der, C.J., Punke, S.G., and Buss, J.E. (1999). A non-farnesylated Ha-Ras protein can be palmitoylated and trigger potent differentiation and transformation. J Biol Chem 274, 1423-1431.

Bos, J.L. (1998). All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral. EMBO J 17, 6776-6782.

Bos, J.L., de Rooij, J., and Reedquist, K.A. (2001). Rap1 signalling: adhering to new models. Nat Rev Mol Cell Biol 2, 369-377.

Bose, I., Irazoqui, J.E., Moskow, J.J., Bardes, E.S., Zyla, T.R., and Lew, D.J. (2001). Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p. J Biol Chem 276, 7176-7186.

Bourne, H.R., Sanders, D.A., and McCormick, F. (1990). The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125-132.

Butty, A.C., Perrinjaquet, N., Petit, A., Jaquenoud, M., Segall, J.E., Hofmann, K., Zwahlen, C., and Peter, M. (2002). A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization. EMBO J 21, 1565-1576.

Casamayor, A., and Snyder, M. (2002). Bud-site selection and cell polarity in budding yeast. Curr Opin Microbiol 5, 179-186.

Casey, P.J., and Seabra, M.C. (1996). Protein prenyltransferases. J Biol Chem 271, 5289-5292.

Chant, J. (1999). Cell polarity in yeast. Annu Rev Cell Dev Biol 15, 365-391.

Chant, J., Corrado, K., Pringle, J.R., and Herskowitz, I. (1991). Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for bud site selection and interacts with bud formation gene BEM1. Cell 65, 1213-1224.

Chant, J., Mischke, M., Mitchell, E., Herskowitz, I., and Pringle, J.R. (1995). Role of Bud3p in producing the axial budding pattern of yeast. J Cell Biol 129, 767-778.

Chavrier, P., and Menetrey, J. (2010). Toward a structural understanding of arf family:effector specificity. Structure 18, 1552-1558.

Chen, K.Y., Tsai, P.C., Hsu, J.W., Hsu, H.C., Fang, C.Y., Chang, L.C., Tsai, Y.T., Yu, C.J., and Lee, F.J. (2010). Syt1p promotes activation of Arl1p at the late Golgi to recruit Imh1p. J Cell Sci 123, 3478-3489.

Chesneau, L., Dambournet, D., Machicoane, M., Kouranti, I., Fukuda, M., Goud, B., and Echard, A. (2012). An ARF6/Rab35 GTPase cascade for endocytic recycling and successful cytokinesis. Curr Biol 22, 147-153.

Costa, R., and Ayscough, K.R. (2005). Interactions between Sla1p, Lsb5p and Arf3p in yeast endocytosis. Biochem Soc Trans 33, 1273-1275.

Costa, R., Warren, D.T., and Ayscough, K.R. (2005). Lsb5p interacts with actin regulators Sla1p and Las17p, ubiquitin and Arf3p to couple actin dynamics to membrane trafficking processes. Biochem J 387, 649-658.

Cotton, M., Boulay, P.L., Houndolo, T., Vitale, N., Pitcher, J.A., and Claing, A. (2007). Endogenous ARF6 interacts with Rac1 upon angiotensin II stimulation to regulate membrane ruffling and cell migration. Mol Biol Cell 18, 501-511.

Cullen, P.J., Sabbagh, W., Jr., Graham, E., Irick, M.M., van Olden, E.K., Neal, C., Delrow, J., Bardwell, L., and Sprague, G.F., Jr. (2004). A signaling mucin at the head of the Cdc42- and MAPK-dependent filamentous growth pathway in yeast. Genes Dev 18, 1695-1708.

Cullen, P.J., and Sprague, G.F., Jr. (2000). Glucose depletion causes haploid invasive growth in yeast. Proc Natl Acad Sci U S A 97, 13619-13624.

Cullen, P.J., and Sprague, G.F., Jr. (2002). The roles of bud-site-selection proteins during haploid invasive growth in yeast. Mol Biol Cell 13, 2990-3004.
Cullen, P.J., and Sprague, G.F., Jr. (2012). The regulation of filamentous growth in yeast. Genetics 190, 23-49.

D''Souza-Schorey, C., and Chavrier, P. (2006). ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 7, 347-358.

De Matteis, M.A., and Luini, A. (2008). Exiting the Golgi complex. Nat Rev Mol Cell Biol 9, 273-284.

Donaldson, J.G., and Jackson, C.L. (2011). ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat Rev Mol Cell Biol 12, 362-375.

Drubin, D.G. (1991). Development of cell polarity in budding yeast. Cell 65, 1093-1096.

Drubin, D.G., and Nelson, W.J. (1996). Origins of cell polarity. Cell 84, 335-344.

Enomoto, K., and Gill, D.M. (1980). Cholera toxin activation of adenylate cyclase. Roles of nucleoside triphosphates and a macromolecular factor in the ADP ribosylation of the GTP-dependent regulatory component. J Biol Chem 255, 1252-1258.

Fichtner, L., Schulze, F., and Braus, G.H. (2007). Differential Flo8p-dependent regulation of FLO1 and FLO11 for cell-cell and cell-substrate adherence of S. cerevisiae S288c. Mol Microbiol 66, 1276-1289.

Garcia-Ranea, J.A., and Valencia, A. (1998). Distribution and functional diversification of the ras superfamily in Saccharomyces cerevisiae. FEBS Lett 434, 219-225.

Gietz, R.D., and Sugino, A. (1988). New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527-534.

Gillingham, A.K., and Munro, S. (2007a). Identification of a guanine nucleotide exchange factor for Arf3, the yeast orthologue of mammalian Arf6. PLoS One 2, e842.

Gillingham, A.K., and Munro, S. (2007b). The small G proteins of the Arf family and their regulators. Annu Rev Cell Dev Biol 23, 579-611.

Gimeno, C.J., and Fink, G.R. (1992). The logic of cell division in the life cycle of yeast. Science 257, 626.

Gimeno, C.J., Ljungdahl, P.O., Styles, C.A., and Fink, G.R. (1992). Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68, 1077-1090.

Gladfelter, A.S., Bose, I., Zyla, T.R., Bardes, E.S., and Lew, D.J. (2002). Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p. J Cell Biol 156, 315-326.

Glomset, J.A., and Farnsworth, C.C. (1994). Role of protein modification reactions in programming interactions between ras-related GTPases and cell membranes. Annu Rev Cell Biol 10, 181-205.

Graham, T.R. (2004). Membrane targeting: getting Arl to the Golgi. Curr Biol 14, R483-485.

Guldal, C.G., and Broach, J. (2006). Assay for adhesion and agar invasion in S. cerevisiae. J Vis Exp, 64.

Guo, B., Styles, C.A., Feng, Q., and Fink, G.R. (2000). A Saccharomyces gene family involved in invasive growth, cell-cell adhesion, and mating. Proc Natl Acad Sci U S A 97, 12158-12163.

Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993). Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75, 791-803.

Hall, A. (1990). The cellular functions of small GTP-binding proteins. Science 249, 635-640.

Halme, A., Michelitch, M., Mitchell, E.L., and Chant, J. (1996). Bud10p directs axial cell polarization in budding yeast and resembles a transmembrane receptor. Curr Biol 6, 570-579.

Hausauer, D.L., Gerami-Nejad, M., Kistler-Anderson, C., and Gale, C.A. (2005). Hyphal guidance and invasive growth in Candida albicans require the Ras-like GTPase Rsr1p and its GTPase-activating protein Bud2p. Eukaryot Cell 4, 1273-1286.

Hazbun, T.R., Malmstrom, L., Anderson, S., Graczyk, B.J., Fox, B., Riffle, M., Sundin, B.A., Aranda, J.D., McDonald, W.H., Chiu, C.H., Snydsman, B.E., Bradley, P., Muller, E.G., Fields, S., Baker, D., Yates, J.R., 3rd, and Davis, T.N. (2003). Assigning function to yeast proteins by integration of technologies. Mol Cell 12, 1353-1365.

Herskowitz, I. (1988). Life cycle of the budding yeast Saccharomyces cerevisiae. Microbiol Rev 52, 536-553.

Herskowitz, I., Park, H.O., Sanders, S., Valtz, N., and Peter, M. (1995). Programming of cell polarity in budding yeast by endogenous and exogenous signals. Cold Spring Harb Symp Quant Biol 60, 717-727.

Houk, A.R., Millius, A., and Weiner, O.D. (2009). Compete globally, bud locally. Cell 139, 656-658.

Howell, A.S., and Lew, D.J. (2012). Morphogenesis and the cell cycle. Genetics 190, 51-77.

Huang, C.F., Liu, Y.W., Tung, L., Lin, C.H., and Lee, F.J. (2003). Role for Arf3p in development of polarity, but not endocytosis, in Saccharomyces cerevisiae. Mol Biol Cell 14, 3834-3847.

Iden, S., and Collard, J.G. (2008). Crosstalk between small GTPases and polarity proteins in cell polarization. Nat Rev Mol Cell Biol 9, 846-859.

Irazoqui, J.E., Gladfelter, A.S., and Lew, D.J. (2003). Scaffold-mediated symmetry breaking by Cdc42p. Nat Cell Biol 5, 1062-1070.

Irazoqui, J.E., and Lew, D.J. (2004). Polarity establishment in yeast. J Cell Sci 117, 2169-2171.

Jackson, C.L., and Casanova, J.E. (2000). Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors. Trends Cell Biol 10, 60-67.

Jin, R., Dobry, C.J., McCown, P.J., and Kumar, A. (2008). Large-scale analysis of yeast filamentous growth by systematic gene disruption and overexpression. Mol Biol Cell 19, 284-296.

Johnson, D.I. (1999). Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity. Microbiol Mol Biol Rev 63, 54-105.

Kahn, R.A., Cherfils, J., Elias, M., Lovering, R.C., Munro, S., and Schurmann, A. (2006). Nomenclature for the human Arf family of GTP-binding proteins: ARF, ARL, and SAR proteins. J Cell Biol 172, 645-650.

Kang, P.J., Beven, L., Hariharan, S., and Park, H.O. (2010). The Rsr1/Bud1 GTPase interacts with itself and the Cdc42 GTPase during bud-site selection and polarity establishment in budding yeast. Mol Biol Cell 21, 3007-3016.

Kang, P.J., Sanson, A., Lee, B., and Park, H.O. (2001). A GDP/GTP exchange factor involved in linking a spatial landmark to cell polarity. Science 292, 1376-1378.

Kotani, K., Kikuchi, A., Doi, K., Kishida, S., Sakoda, T., Kishi, K., and Takai, Y. (1992). The functional domain of the stimulatory GDP/GTP exchange protein (smg GDS) which interacts with the C-terminal geranylgeranylated region of rap1/Krev-1/smg p21. Oncogene 7, 1699-1704.

Kozminski, K.G., Beven, L., Angerman, E., Tong, A.H., Boone, C., and Park, H.O. (2003). Interaction between a Ras and a Rho GTPase couples selection of a growth site to the development of cell polarity in yeast. Mol Biol Cell 14, 4958-4970.

Kozubowski, L., Saito, K., Johnson, J.M., Howell, A.S., Zyla, T.R., and Lew, D.J. (2008). Symmetry-breaking polarization driven by a Cdc42p GEF-PAK complex. Curr Biol 18, 1719-1726.

Kron, S.J. (1997). Filamentous growth in budding yeast. Trends Microbiol 5, 450-454.

Kuchin, S., Vyas, V.K., and Carlson, M. (2002). Snf1 protein kinase and the repressors Nrg1 and Nrg2 regulate FLO11, haploid invasive growth, and diploid pseudohyphal differentiation. Mol Cell Biol 22, 3994-4000.

Kupzig, S., Deaconescu, D., Bouyoucef, D., Walker, S.A., Liu, Q., Polte, C.L., Daumke, O., Ishizaki, T., Lockyer, P.J., Wittinghofer, A., and Cullen, P.J. (2006). GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins. J Biol Chem 281, 9891-9900.

Lambert, A.A., Perron, M.P., Lavoie, E., and Pallotta, D. (2007). The Saccharomyces cerevisiae Arf3 protein is involved in actin cable and cortical patch formation. FEMS Yeast Res 7, 782-795.

Lee, F.J., Stevens, L.A., Kao, Y.L., Moss, J., and Vaughan, M. (1994). Characterization of a glucose-repressible ADP-ribosylation factor 3 (ARF3) from Saccharomyces cerevisiae. J Biol Chem 269, 20931-20937.

Lemaire, K., Van de Velde, S., Van Dijck, P., and Thevelein, J.M. (2004). Glucose and sucrose act as agonist and mannose as antagonist ligands of the G protein-coupled receptor Gpr1 in the yeast Saccharomyces cerevisiae. Mol Cell 16, 293-299.

Liu, H., Styles, C.A., and Fink, G.R. (1993). Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science 262, 1741-1744.

Liu, H., Styles, C.A., and Fink, G.R. (1996). Saccharomyces cerevisiae S288C has a mutation in FLO8, a gene required for filamentous growth. Genetics 144, 967-978.

Liu, Y.W., Lee, S.W., and Lee, F.J. (2006). Arl1p is involved in transport of the GPI-anchored protein Gas1p from the late Golgi to the plasma membrane. J Cell Sci 119, 3845-3855.

Lo, W.S., and Dranginis, A.M. (1998). The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae. Mol Biol Cell 9, 161-171.

Lo, W.S., Raitses, E.I., and Dranginis, A.M. (1997). Development of pseudohyphae by embedded haploid and diploid yeast. Curr Genet 32, 197-202.

Longtine, M.S., and Bi, E. (2003). Regulation of septin organization and function in yeast. Trends Cell Biol 13, 403-409.

Longtine, M.S., McKenzie, A., 3rd, Demarini, D.J., Shah, N.G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J.R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953-961.

Lorenz, M.C., Pan, X., Harashima, T., Cardenas, M.E., Xue, Y., Hirsch, J.P., and Heitman, J. (2000). The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Genetics 154, 609-622.

Macia, E., Chabre, M., and Franco, M. (2001). Specificities for the small G proteins ARF1 and ARF6 of the guanine nucleotide exchange factors ARNO and EFA6. J Biol Chem 276, 24925-24930.

Magee, A.I., Newman, C.M., Giannakouros, T., Hancock, J.F., Fawell, E., and Armstrong, J. (1992). Lipid modifications and function of the ras superfamily of proteins. Biochem Soc Trans 20, 497-499.

Maidan, M.M., De Rop, L., Serneels, J., Exler, S., Rupp, S., Tournu, H., Thevelein, J.M., and Van Dijck, P. (2005). The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMP-protein kinase A pathway to induce morphogenesis in Candida albicans. Mol Biol Cell 16, 1971-1986.

Michelitch, M., and Chant, J. (1996). A mechanism of Bud1p GTPase action suggested by mutational analysis and immunolocalization. Curr Biol 6, 446-454.

Mosch, H.U., and Fink, G.R. (1997). Dissection of filamentous growth by transposon mutagenesis in Saccharomyces cerevisiae. Genetics 145, 671-684.

Mosch, H.U., Roberts, R.L., and Fink, G.R. (1996). Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 93, 5352-5356.

Moss, J., and Vaughan, M. (1995). Structure and function of ARF proteins: activators of cholera toxin and critical components of intracellular vesicular transport processes. J Biol Chem 270, 12327-12330.

Nazarko, V.Y., Thevelein, J.M., and Sibirny, A.A. (2008). G-protein-coupled receptor Gpr1 and G-protein Gpa2 of cAMP-dependent signaling pathway are involved in glucose-induced pexophagy in the yeast Saccharomyces cerevisiae. Cell Biol Int 32, 502-504.

Osmani, N., Peglion, F., Chavrier, P., and Etienne-Manneville, S. (2010). Cdc42 localization and cell polarity depend on membrane traffic. J Cell Biol 191, 1261-1269.

Palecek, S.P., Parikh, A.S., and Kron, S.J. (2002). Sensing, signalling and integrating physical processes during Saccharomyces cerevisiae invasive and filamentous growth. Microbiology 148, 893-907.

Palmieri, S.J., and Haarer, B.K. (1998). Polarity and division site specification in yeast. Curr Opin Microbiol 1, 678-686.

Pan, X., Harashima, T., and Heitman, J. (2000). Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae. Curr Opin Microbiol 3, 567-572.

Panic, B., Whyte, J.R., and Munro, S. (2003). The ARF-like GTPases Arl1p and Arl3p act in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus. Curr Biol 13, 405-410.

Park, H.O., and Bi, E. (2007). Central roles of small GTPases in the development of cell polarity in yeast and beyond. Microbiol Mol Biol Rev 71, 48-96.

Park, H.O., Bi, E., Pringle, J.R., and Herskowitz, I. (1997). Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity. Proc Natl Acad Sci U S A 94, 4463-4468.

Park, H.O., Chant, J., and Herskowitz, I. (1993). BUD2 encodes a GTPase-activating protein for Bud1/Rsr1 necessary for proper bud-site selection in yeast. Nature 365, 269-274.

Park, H.O., Kang, P.J., and Rachfal, A.W. (2002). Localization of the Rsr1/Bud1 GTPase involved in selection of a proper growth site in yeast. J Biol Chem 277, 26721-26724.

Park, H.O., Sanson, A., and Herskowitz, I. (1999). Localization of Bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site. Genes Dev 13, 1912-1917.

Perez, P., and Rincon, S.A. (2010). Rho GTPases: regulation of cell polarity and growth in yeasts. Biochem J 426, 243-253.

Pruyne, D., and Bretscher, A. (2000a). Polarization of cell growth in yeast. J Cell Sci 113 ( Pt 4), 571-585.

Pruyne, D., and Bretscher, A. (2000b). Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J Cell Sci 113 ( Pt 3), 365-375.

Radhakrishna, H., Al-Awar, O., Khachikian, Z., and Donaldson, J.G. (1999). ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements. J Cell Sci 112 ( Pt 6), 855-866.

Radhakrishna, H., and Donaldson, J.G. (1997). ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway. J Cell Biol 139, 49-61.

Randazzo, P.A., Nie, Z., Miura, K., and Hsu, V.W. (2000). Molecular aspects of the cellular activities of ADP-ribosylation factors. Sci STKE 2000, re1.

Rivera-Molina, F.E., and Novick, P.J. (2009). A Rab GAP cascade defines the boundary between two Rab GTPases on the secretory pathway. Proc Natl Acad Sci U S A 106, 14408-14413.

Roemer, T., Madden, K., Chang, J., and Snyder, M. (1996). Selection of axial growth sites in yeast requires Axl2p, a novel plasma membrane glycoprotein. Genes Dev 10, 777-793.

Rolland, F., De Winde, J.H., Lemaire, K., Boles, E., Thevelein, J.M., and Winderickx, J. (2000). Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process. Mol Microbiol 38, 348-358.

Ruggieri, R., Bender, A., Matsui, Y., Powers, S., Takai, Y., Pringle, J.R., and Matsumoto, K. (1992). RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae. Mol Cell Biol 12, 758-766.

Sabe, H. (2003). Requirement for Arf6 in cell adhesion, migration, and cancer cell invasion. J Biochem 134, 485-489.

Sanders, S.L., and Herskowitz, I. (1996). The BUD4 protein of yeast, required for axial budding, is localized to the mother/BUD neck in a cell cycle-dependent manner. J Cell Biol 134, 413-427.

Shimada, Y., Gulli, M.P., and Peter, M. (2000). Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating. Nat Cell Biol 2, 117-124.

Shimada, Y., Wiget, P., Gulli, M.P., Bi, E., and Peter, M. (2004). The nucleotide exchange factor Cdc24p may be regulated by auto-inhibition. EMBO J 23, 1051-1062.

Sikorski, R.S., and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27.

Singer-Kruger, B., Lasic, M., Burger, A.M., Hausser, A., Pipkorn, R., and Wang, Y. (2008). Yeast and human Ysl2p/hMon2 interact with Gga adaptors and mediate their subcellular distribution. EMBO J 27, 1423-1435.

Smaczynska-de, R., II, Costa, R., and Ayscough, K.R. (2008). Yeast Arf3p modulates plasma membrane PtdIns(4,5)P2 levels to facilitate endocytosis. Traffic 9, 559-573.

Song, J., Khachikian, Z., Radhakrishna, H., and Donaldson, J.G. (1998). Localization of endogenous ARF6 to sites of cortical actin rearrangement and involvement of ARF6 in cell spreading. J Cell Sci 111 ( Pt 15), 2257-2267.

Spang, A., Matsuoka, K., Hamamoto, S., Schekman, R., and Orci, L. (1998). Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes. Proc Natl Acad Sci U S A 95, 11199-11204.

Sudbery, P.E. (2011). Growth of Candida albicans hyphae. Nat Rev Microbiol 9, 737-748.

Takai, Y., Sasaki, T., and Matozaki, T. (2001). Small GTP-binding proteins. Physiol Rev 81, 153-208.

Tamaki, H., Miwa, T., Shinozaki, M., Saito, M., Yun, C.W., Yamamoto, K., and Kumagai, H. (2000). GPR1 regulates filamentous growth through FLO11 in yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 267, 164-168.

Thevelein, J.M., and de Winde, J.H. (1999). Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol 33, 904-918.

Thompson, B.J. (2012). Cell polarity: models and mechanisms from yeast, worms and flies. Development 140, 13-21.

Toret, C.P., Lee, L., Sekiya-Kawasaki, M., and Drubin, D.G. (2008). Multiple pathways regulate endocytic coat disassembly in Saccharomyces cerevisiae for optimal downstream trafficking. Traffic 9, 848-859.

Tsai, P.C., Hsu, J.W., Liu, Y.W., Chen, K.Y., and Lee, F.J. (2013). Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p. Proc Natl Acad Sci U S A 110, E668-677.

Tsai, P.C., Lee, S.W., Liu, Y.W., Chu, C.W., Chen, K.Y., Ho, J.C., and Lee, F.J. (2008). Afi1p functions as an Arf3p polarization-specific docking factor for development of polarity. J Biol Chem 283, 16915-16927.

Vernet, T., Dignard, D., and Thomas, D.Y. (1987). A family of yeast expression vectors containing the phage f1 intergenic region. Gene 52, 225-233.

Verstrepen, K.J., Reynolds, T.B., and Fink, G.R. (2004). Origins of variation in the fungal cell surface. Nat Rev Microbiol 2, 533-540.

Vigil, D., Cherfils, J., Rossman, K.L., and Der, C.J. (2010). Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? Nat Rev Cancer 10, 842-857.

Vyas, V.K., Kuchin, S., Berkey, C.D., and Carlson, M. (2003). Snf1 kinases with different beta-subunit isoforms play distinct roles in regulating haploid invasive growth. Mol Cell Biol 23, 1341-1348.

Wu, X., and Jiang, Y.W. (2005). Genetic/genomic evidence for a key role of polarized endocytosis in filamentous differentiation of S. cerevisiae. Yeast 22, 1143-1153.

Zakrzewska, E., Perron, M., Laroche, A., and Pallotta, D. (2003). A role for GEA1 and GEA2 in the organization of the actin cytoskeleton in Saccharomyces cerevisiae. Genetics 165, 985-995.

Zhang, F.L., and Casey, P.J. (1996). Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem 65, 241-269.

Zhu, Y., Drake, M.T., and Kornfeld, S. (1999). ADP-ribosylation factor 1 dependent clathrin-coat assembly on synthetic liposomes. Proc Natl Acad Sci U S A 96, 5013-5018.

Zhu, Y., Drake, M.T., and Kornfeld, S. (2001). Adaptor protein 1-dependent clathrin coat assembly on synthetic liposomes and Golgi membranes. Methods Enzymol 329, 379-387.