Sip1, an AP-1 Accessory Protein in Fission Yeast, Is Required for Localization of Rho3 GTPase

Abstract

Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3 + suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of Golgi/endosomal trafficking in fission yeast.

Rho4 interaction with exocyst and septins regulates cell separation in fission yeast

“…Rho3 binds to different subunits of the AP-1 complex, which mediates transport from Golgi to endosomes. Thus, Rho3 interacts with the AP-1 accessory protein Sip1 in a nucleotideindependent way, but interacts with Apm1 preferentially in its active form (Kita et al, 2011;Yu et al, 2013). Taken together, these data indicate that exocyst localization might be precisely regulated in time and space and several GTPases such as Cdc42, Rho4 and Rho3 are involved in this process.…”

A role for F-BAR protein Rga7p during cytokinesis in S. pombe

“…For3p assembles actin filament cables during interphase (Feierbach and Chang, 2001;Martin and Chang, 2006) but also localizes to the cleavage furrow where it contributes to the timely assembly and constriction of contractile rings (Coffman et al, 2013). For3p binds to activated Rho3p and Cdc42p, both of which are involved in membrane trafficking (Estravis et al, 2011;Martin et al, 2007;Nakano et al, 2002), and Rho3p localizes to the Golgi and endosomes, as well as to the division site (Yu et al, 2013). Furthermore, the synthetic lethal genetic interactions of both Δrga7 and Δfor3 with mutations of the genes for myosin-II essential light chain cdc4 + and IQGAP rng2 + (Table 1) (Coffman et al, 2013) are likely to result from the combined defects in the contractile ring assembly and septum assembly.…”

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

“…In S. pombe, mutations in exocyst components show defects in cell separation (28,31,32). Rho3p restored exocyst localization in a cdc42 thermosensitive mutant with multiple membrane traffic defects (33), and it has been implicated in the regulation of Golgi/endosome trafficking through a functional interaction with adaptin (clathrin-associated adaptor protein-1) and Sip1p (34,35). Thus, it is possible that Rho3p could stimulate secretion by locally increasing the exocytic apparatus or through Golgi/endosome regulation.…”