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

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.

Endosomal cargo recycling mediated by Gpa1 and phosphatidylinositol 3-kinase is inhibited by glucose starvation

“…We note that disruption of these regulators does not exhibit the extreme FM4-64 recycling defects found in glucose-starved ( Figure 1D ) or PI3K-null ( Figure 4C ) cells, but it is similar to deletion of other factors previously shown to perturb recycling, such as Rcy1, the Rag GTPases, and the Rpd3 complex ( Wiederkehr et al. , 2000 ; MacDonald and Piper, 2017 ; Amoiradaki et al. , 2021 ).…”

“…, 2021 ). Genetic dissection of the latter pathway implies that recycling is controlled at transcriptional and metabolic levels ( MacDonald and Piper, 2017 ; Amoiradaki et al. , 2021 ), suggesting that early endocytic trafficking decisions contribute to the eventual down-regulation of surface cargoes in response to nutritional cues.…”

The phosphatase Glc7 controls the eisosomal response to starvation via post-translational modification of Pil1

“…Since then it has been found to have many functional associations with nutrient signalling ( Conrad et al, 2014 ). Sit4 has also been implicated in trafficking pathways used by nutrient transporters at the endoplasmic reticulum ( Bhandari et al, 2013 ) and from endosomes to the surface ( Amoiradaki et al, 2021 ) and vacuole ( Gurunathan et al, 2002 ). Yvh1 is a phospho-tyrosine-specific enzyme ( Guan et al, 1992 ) that is mainly associated with ribosome maturation ( Kemmler et al, 2009 ) but has also been implicated in autophagy downstream of TORC1 following nutrient depletion ( Yeasmin et al, 2015 ).…”

The histone deacetylases Rpd3 and Hst1 antagonistically regulate de novo NAD+ metabolism in the budding yeast Saccharomyces cerevisiae

“…Rpd3 has also been implicated in recycling of Tat2, a TRP transporter. The growth of a strain defective in Tat2-depedent transport is significantly inhibited under low TRP conditions by deletion of RPD3 ( 59 ). We saw, however, that rpd3Δ cells remain proficient for TRP uptake, accumulating the metabolite above levels seen in WT cells ( Fig.…”

“…3 A ). This suggests that reduced assimilation of TRP into NAD + via the de novo pathway may contribute to the growth defects of rpd3Δ cells in low TRP media ( 59 ). In addition to defective de novo NAD + metabolism, cells lacking Rpd3 also show altered NA–NAM and NR salvage activities.…”