The Rpd3-Complex Regulates Expression of Multiple Cell Surface Recycling Factors in Yeast.

Konstantina Amoiradaki,Chris Macdonald,Josephine E Ayre,Kate R Bunting,Kamilla M E Laidlaw,Karen Hogg,Katherine M Paine

doi:10.3390/ijms222212477

Konstantina Amoiradaki, Chris Macdonald + Show 5 more

Open Access

https://doi.org/10.3390/ijms222212477

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Abstract

Intracellular trafficking pathways control residency and bioactivity of integral membrane proteins at the cell surface. Upon internalisation, surface cargo proteins can be delivered back to the plasma membrane via endosomal recycling pathways. Recycling is thought to be controlled at the metabolic and transcriptional level, but such mechanisms are not fully understood. In yeast, recycling of surface proteins can be triggered by cargo deubiquitination and a series of molecular factors have been implicated in this trafficking. In this study, we follow up on the observation that many subunits of the Rpd3 lysine deacetylase complex are required for recycling. We validate ten Rpd3-complex subunits in recycling using two distinct assays and developed tools to quantify both. Fluorescently labelled Rpd3 localises to the nucleus and complements recycling defects, which we hypothesised were mediated by modulated expression of Rpd3 target gene(s). Bioinformatics implicated 32 candidates that function downstream of Rpd3, which were over-expressed and assessed for capacity to suppress recycling defects of rpd3∆ cells. This effort yielded three hits: Sit4, Dit1 and Ldb7, which were validated with a lipid dye recycling assay. Additionally, the essential phosphatidylinositol-4-kinase Pik1 was shown to have a role in recycling. We propose recycling is governed by Rpd3 at the transcriptional level via multiple downstream target genes.

Highlights

Most integral membrane proteins expressed in eukaryotic cells are inserted into the endoplasmic reticulum (ER) via different mechanisms [1]
We stably integrated Ste3-GFP-deubiquitinating enzyme (DUb) into wild-type cells and all 10 previous identified mutants of the Rpd3 complex, revealing all mutants had some degree of recycling defect, with intracellular accumulation of reporter similar to that observed in rcy1∆ cells (Figure 1C)
To quantify differences between the null strains lacking components of the Rpd3-complex, we optimised the segmentation of cells using phase contrast (PC) and digital interference contrast (DIC)

Summary

Introduction

Most integral membrane proteins expressed in eukaryotic cells are inserted into the endoplasmic reticulum (ER) via different mechanisms [1]. Many of these perform diverse roles at the plasma membrane (PM), such as acting as ion channels, nutrient transporters, and different classes of receptors [2,3]. Inactive nutrient transporters localised to eisosome subdomains adopt an active conformation for nutrient uptake upon migration to other regions of the PM in response to a substrate [7,8]. This altered PM localisation of active transporters supports their internalisation and endocytosis [9], a process which is controlled metabolically, with stress conditions altering eisosomal capacity to harbour nutrient transporters [10,11]

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