Role of the Small GTPase Rho3 in Golgi/Endosome Trafficking through Functional Interaction with Adaptin in Fission Yeast

Abstract

BackgroundWe had previously identified the mutant allele of apm1+ that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast.Methodology/Principal FindingsIn the present study, we isolated rho3+, which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl− sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl−, and valproic acid. Green fluorescent protein (GFP)-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner.Conclusions/SignificanceTaken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence of a direct link between the small GTPase Rho and the clathrin-associated adaptor protein-1 in membrane trafficking.

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.…”

“…6c). It is known that rho3D accumulate abnormal electrodense membranes inside the cells and have secretion defects (Kita et al, 2011), so it is possible that Exo70 is retained in these internal membranes. In summary, rho3D rho4D double mutant displayed additive effects (Fig.…”

“…rho3 + overexpression also suppresses the growth defect of cdc42 mutant alleles with various membrane traffic defects (Estravís et al, 2011). Additionally, Rho3 participates in Golgi/endosome trafficking through interaction with Apm1, a protein of the clathrin-associated adaptor complex AP-1 (Kita et al, 2011). Rho3 and/or Rho4 orthologues have been described in other fungi, such as Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, and Aspergillus nidulans (Elias & Klimes, 2012). S. cerevisiae Rho3 and Rho4 are partially redundant, and are involved in cell polarity through regulation of polarized exocytosis and actin cytoskeleton organization (Matsui & Toh-E, 1992b).…”

Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

“…8D). Rho3 is involved in polarized cell growth by regulating proper localization of formin For3 and the exocyst (58,59) and in the control of Golgi-endosome trafficking (60). Interestingly, increased expression of Erf2/Erf4 enhances the palmitoylation state of Rho3 to promote meiotic entry (49).…”

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.…”