Sip1, a Conserved AP-1 Accessory Protein, Is Important for Golgi/Endosome Trafficking in Fission Yeast

Masako Udo,Nanae Umeda,Kanako Hagihara,Ayako Kita,Mami Shintani,Yang Yu,Juan Mata,Yuta Katayama,Kwihwa Park,Reiko Sugiura

doi:10.1371/journal.pone.0045324

Abstract

We had previously identified the mutant allele of apm1+ that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4+/sip1+, which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1 -deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl− and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking.

Highlights

Clathrin adaptor protein (AP) complexes play a key role in the transport of proteins by regulating the formation of transport vesicles as well as cargo selection between organelles of the postGolgi network, namely the trans-Golgi network, endosomes, lysosomes, and the plasma membrane [1]
Homolog of the Clathrin-associated Adaptor Protein-1 (AP-1) Complex-Interacting Protein The its4+ gene was cloned by complementation of the temperature-sensitive growth defect of its4-1 mutant cells (Figure 1A, sip1-i4+its4+). its4+ complemented sensitivities to drugs associated with the its4-1 mutant (Figure 1A)
We present several lines of evidence that suggest a role for Sip1 in Golgi/endosomal trafficking as an adaptor protein-1 (AP-1) accessory protein

Summary

Introduction

Clathrin adaptor protein (AP) complexes play a key role in the transport of proteins by regulating the formation of transport vesicles as well as cargo selection between organelles of the postGolgi network, namely the trans-Golgi network, endosomes, lysosomes, and the plasma membrane [1]. Accessory proteins that bind to the AP complex have been attracting increasing attention for their role in membrane trafficking as well as in signaling pathways [4,5]. Compared with AP-2-binding proteins, less is known about AP-1 accessories with the exceptions of c-synergin [6], Eps15 [7], epsinR [8], and p200 [9]. C-Synergin is associated with AP-1 both in the cytosol and on trans-Golgi network (TGN) membranes. It binds directly to the ear domain of c-adaptin and contains an Eps homology (EH) domain, the EH domain is not part of the c-adaptin binding site

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