Ent3 and GGA adaptors facilitate diverse anterograde and retrograde trafficking events to and from the prevacuolar endosome

Francisco Yanguas,Esteban Moscoso-Romero,M.-Henar Valdivieso

doi:10.1038/s41598-019-47035-5

Abstract

Carboxypeptidases Y (Cpy1) and S (Cps1), the receptor Vps10, and the ATPase subunit Vph1 follow the carboxypeptidase Y (CPY) pathway from the trans-Golgi network (TGN) to the prevacuolar endosome (PVE). Using Schizosaccharomyces pombe quantitative live-cell imaging, biochemical and genetic analyses, we extended the previous knowledge and showed that collaboration between Gga22, the dominant Golgi-localized Gamma-ear-containing ARF-binding (GGA) protein, and Gga21, and between Gga22 and the endosomal epsin Ent3, was required for efficient: i) Vps10 anterograde trafficking from the TGN to the PVE; ii) Vps10 retrograde trafficking from the PVE to the TGN; iii) Cps1 exit from the TGN, and its sorting in the PVE en route to the vacuole; and iv) Syb1/Snc1 recycling to the plasma membrane through the PVE. Therefore, monomeric clathrin adaptors facilitated the trafficking of Vps10 in both directions of the CPY pathway, and facilitated trafficking events of Cps1 in different organelles. By contrast, they were dispensable for Vph1 trafficking. Thus, these adaptors regulated the traffic of some, but not all, of the cargo of the CPY pathway, and regulated the traffic of cargoes that do not follow this pathway. Additionally, this collaboration was required for PVE organization and efficient growth under stress.

Highlights

Vacuolar hydrolases, such as the carboxypeptidase Y (Prc1/Cpy1), proteinase A, and carboxypeptidase S (Cps1) are synthesized in the endoplasmic reticulum, before being transported to the TGN
We performed colocalization studies using the exomer component Cfr1-RFP, which acted as a stable TGN marker (Fig. S1), and the phosphatidylinositol 3-phosphate (PI3P) probe Cherry-FYVE as a PVE marker[2,30]
The fact that the S. pombe receptor was observed at the vacuole membrane in retromer mutants[8], showed that in fission yeast Vps10-green fluorescent protein (GFP) cycles between the TGN and the PVE, as described in budding yeast[9]

Summary

Introduction

Vacuolar hydrolases, such as the carboxypeptidase Y (Prc1/Cpy1), proteinase A, and carboxypeptidase S (Cps1) are synthesized in the endoplasmic reticulum, before being transported to the TGN There, they are diverted from the general secretory pathway to the endocytic pathway by transmembrane receptors and soluble adaptors (reviewed in[3,4,5,6,7]). Vps[10] is the receptor that interacts with Cpy[1] and proteinase A, which are soluble proteins, ensuring their sorting into clathrin-coated vesicles destined for the PVE. The sorting of transmembrane receptors that follow the CPY pathway requires the participation of Golgi-localized Gamma-ear-containing ARF-binding (GGA) clathrin adaptors[12,13,14,15,16]. We showed that the integrity of the PVE and efficient cell growth under stress conditions depend on collaboration between monomeric clathrin adaptors

Methods

Results

Conclusion