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.

Highlights

  • In eukaryotic cells, Rho family small GTPases play a crucial role in polarized growth through reorganization of the actin cytoskeleton and the regulation of secretory vesicle transport [1]

  • Conclusions/Significance: Taken 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

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Summary

Introduction

Rho family small GTPases play a crucial role in polarized growth through reorganization of the actin cytoskeleton and the regulation of secretory vesicle transport [1]. In the budding yeast Saccharomyces cerevisiae, Rho appears to influence cell growth by regulating polarized secretion as well as the actin cytoskeleton by interacting with Exo and Myo2 [4,5]. In the fission yeast Schizosaccharomyces pombe, Rho is implicated in polarized cell growth through both Formin [7] and by modulating exocyst function [8]. We had previously identified the mutant allele of apm1+ that encodes a homolog of the mammalian m1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex, and we demonstrated the role of Apm in Golgi/ endosome trafficking, secretion, and vacuole fusion in fission yeast

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