Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis.
Bottom Line: Rgf3 is an essential GEF for Rho1 GTPase in fission yeast.Biochemical experiments reveal that the Rgf3 C-terminus binds to Art1.Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site.
Affiliation: Graduate Program of Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH 43210 Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210.Show MeSH
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Mentions: In art1∆ cells, Rgf3 localized to the septal region, but its localization to the mature contractile ring was severely affected (Figure 4A), which suggested that Art1 was important for Rgf3 localization to the contractile ring. However, the Rgf3 levels were also significantly reduced in art1∆ cells (Figure 4, A–C). Thus it was hard to know whether the lack of Rgf3 localization was due to the reduction in overall protein levels. To test whether Art1 plays a direct role in localizing Rgf3, we mislocalized Art1 to various cellular structures using the GFP-binding protein (GBP; Figure 5 and Supplemental Figure S6; Rothbauer et al., 2008). GBP binds to the YFP variant mECitrine but not to cyan fluorescent protein (CFP; Rothbauer et al., 2008; Grallert et al., 2013); we therefore tested whether mislocalized Art1-mECitrine recruited Rgf3-mCFP. We mislocalized Art1 to cortical nodes using anillin Mid1-GBP (Figure 5A) and to SPBs using SIN kinase Cdc7-GBP (Figure 5, B and C). Mislocalized Art1 successfully recruited Rgf3-mCFP to cortical nodes and SPBs (Figure 5, A–C). Only a fraction of Art1 was mislocalized; we observed that Art1 still localized to its native localizations, the constricting ring and the septum (Figure 5, B and C, and Supplemental Figure S6), where Mid1 and Cdc7 are normally absent (Sohrmann et al., 1998; Paoletti and Chang, 2000; Mehta and Gould, 2006). Two lines of evidence indicate the mislocalization is specific: 1) No bleed-through was seen from the YFP to the CFP (440 nm) channel (Supplemental Figure S6A, middle panel); and 2) although Rlc1 is more abundant in cells than Rgf3 (Wu and Pollard, 2005), Rlc1 was not recruited by mislocalized Art1 to SPBs (Supplemental Figure S6B). Together these data indicate that Art1 is important for both the localization and protein levels of Rgf3.
Affiliation: Graduate Program of Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH 43210 Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210.