The Npr1 Kinase Controls Biosynthetic and Endocytic Sorting of the Yeast Gap1 Permease

Abstract

Membrane trafficking of the general amino acid permease (Gap1) of Saccharomyces cerevisiae is under nitrogen regulation. In cells growing on proline or urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates. Upon addition of NH 4 ؉ , a preferential nitrogen source, Gap1 is endocytosed and targeted to the vacuole, where it is degraded. This down-regulation requires ubiquitination of the permease, and this ubiquitination is dependent on the essential Npi1/Rsp5 ubiquitin ligase. In this study, we investigated the role of the Npr1 kinase in the regulation of Gap1 trafficking. We show that Npr1 is required for stabilization of Gap1 at the plasma membrane: when an npr1 ts mutant growing on proline is shifted to the restrictive temperature, Gap1 down-regulation is triggered, as it is when NH 4 ؉ is added to wildtype cells. The fate of newly synthesized Gap1 en route to the plasma membrane is also under Npr1 control: in an npr1⌬ mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane. Similar direct sorting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH 4 ؉ . Finally, Gap1 is phosphorylated in NPR1 cells, but this phosphorylation is not strictly dependent on Npr1. Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 trafficking and that this control is exerted not only on Gap1 present at the plasma membrane but also on Gap1 late in the secretory pathway. Npr1 belongs to a subgroup of protein kinases, some of which are reported to exert a positive control on the activity of other permeases. We propose that these kinases also function as regulators of permease trafficking.

Aneuploidy shortens replicative lifespan in Saccharomyces cerevisiae

“…Instead, damaged membrane proteins are targeted for endocytosis and pass through the multivesicular body (MVB) and to the lysosome (in yeast, the vacuole), where they are eventually degraded (Piper & Luzio, 2007; Zhao et al ., 2013). Bul1 plays an important role in this plasma membrane quality control system by facilitating the Rsp5 ‐ dependent polyubiquitination of multiple protein targets which directs their sorting to the vacuole for degradation (Yashiroda et al ., 1996; De Craene et al ., 2001; Helliwell et al ., 2001; Crespo et al ., 2004; Merhi & André, 2012; O'Donnell, 2012; Zhao et al ., 2013). Disruption of this membrane protein quality control pathway has been previously linked to accelerated aging (Fabrizio et al ., 2010), while the Rsp5/Bul1 node, in particular, has been shown to ameliorate the effects of protein aggregation in a model of neurodegenerative diseases (Tardiff et al ., 2013).…”

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…The protein kinase Npr1 plays a major role in the sorting of these two classes of permeases. Npr1 is required for stabilization of Gap1 at the plasma membrane and induces the degradation of Tat2, possibly by regulating their ubiquitination (Schmidt et al 1998;Springael and Andre 1998;De Craene et al 2001;Helliwell et al 2001;Soetens et al 2001;Springael et al 2002). TORC1 activity, through control of the Tap42-Sit4 phosphatase complex, promotes the phosphorylation of Npr1 (Schmidt et al 1998;Jacinto et al 2001;Gander et al 2008).…”

Permease Recycling and Ubiquitination Status Reveal a Particular Role for Bro1 in the Multivesicular Body Pathway

“…The plasmids used in this study are listed in Table I. Plasmid pJOD10 is identical to pCJ025 (26). Cells were grown in minimal buffered medium (pH 6.1) (39) with 3% glucose as the carbon source except where indicated otherwise.…”