The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease

Membrane trafficking of the general amino acid permease (Gap1) of Saccharom yces cerevisiae is under nitrogen regulation. In cells growing on proline o r urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates. Upon addition of NH4+, a prefere ntial nitrogen source, Gapl is endocy-tosed and targeted to the vacuole, wh ere it is degraded. This down-regulation requires ubiquitination of the per mease, and this ubiquitination is dependent on the essential Npil/Rsp5 ubiq uitin 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 stab ilization of Gap1 at the plasma membrane: when an npr1(t8) mutant growing o n proline is shifted to the restrictive temperature, Gap1 down-regulation i s triggered, as it is when NH4+ is added to wild-type cells. The fate of ne wly synthesized Gap1 en route to the plasma membrane is also under Npr1 con trol: in an npr1 Delta mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane. Similar direct sor ting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH4+. Finally, Gap I is phosphorylated in NPR1 cells, but this pho sphorylation is not strictly dependent on Npr1. Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 traffickin g and that this control is exerted not only on Gap1 present at the plasma m embrane but also on Gap1 late in the secretory pathway. Npr1 belongs to a s ubgroup of protein kinases, some of which are reported to exert a positive control on the activity of other permeases. We propose that these kinases a lso function as regulators of permease trafficking.