Ubiquitin Is Required for Sorting to the Vacuole of the Yeast General Amino Acid Permease, Gap1

Abstract

In yeast, ubiquitin plays a central role in proteolysis of a multitude of proteins and serves also as a signal for endocytosis of many plasma membrane proteins. We showed previously that ubiquitination of the general amino acid permease (Gap1) is essential to its endocytosis followed by vacuolar degradation. These processes occur when NH(4)(+), a preferential source of nitrogen, is added to cells growing on proline or urea, i.e. less favored nitrogen sources. In this study, we show that Gap1 is ubiquitinated on two lysine residues in the cytosolic N terminus (positions 9 and 16). A mutant Gap1 in which both lysines are mutated (Gap1(K9K16)) remains fully stable at the plasma membrane after NH(4)(+) addition. Furthermore, each of the two lysines harbors a poly-ubiquitin chain in which ubiquitin is linked to the lysine 63 of the preceding ubiquitin. The Gap1(K9) and Gap1(K16) mutants, in which a single lysine is mutated, are down-regulated in response to NH(4)(+) although more slowly. In proline-grown cells lacking Npr1, a protein kinase involved in the control of Gap1 trafficking, newly synthesized Gap1 is sorted from the Golgi to the vacuole without passing through the plasma membrane (accompanying article, De Craene, J.-O., Soetens, O., and André, B. (2001) J. Biol. Chem. 276, 43939-43948). We show here that ubiquitination of Gap1 is also required for this direct sorting to the vacuole. In an npr1Delta mutant, neosynthesized Gap1(K9K16) is rerouted to and accumulates at the plasma membrane. Finally, Bul1 and Bul2, two proteins interacting with Npi1/Rsp5, are essential to ubiquitination and down-regulation of cell-surface Gap1, as well as to sorting of neosynthesized Gap1 to the vacuole, as occurs in an npr1Delta mutant. Our results reveal a novel role of ubiquitin in the control of Gap1 trafficking, i.e. direct sorting from the late secretory pathway to the vacuole. This result reinforces the growing evidence that ubiquitin plays an important role not only in internalization of plasma membrane proteins but also in their sorting in the endosomes and/or trans-Golgi.

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter

“…But no Rsp5 plasma membrane endocytic substrates carry such PY motifs. However, Bul1 and Bul2, two PYcontaining proteins, regulate endocytosis of a subset of plasma membrane transporters (7,45,47). Very recently, PY-containing arrestin-like proteins were also shown to play the role of Rsp5-substrate adaptors at the cell surface (5,6).…”

Ubiquitin-Dependent Sorting in Endocytosis

“…Each Art Rsp5 adaptor is thought to have a repertoire of plasma membrane protein cargo that overlaps to various degrees with that of other Art adaptors. For instance, Bul1/2 has a critical role in ubiquitinating and sorting the Gap1 transporter to the vacuole, whereas Art1 plays a critical role in trafficking the Mup1 transporter (Helliwell et al 2001;Soetens et al 2001;Lin et al 2008). In mammalian cells, ARRDC3 controls ubiquitination and down-regulation of b2 and b3 adrenergic receptors as well as the b4 integrin (Draheim et al 2010;Nabhan et al 2010;Patwari et al 2011;Shea et al 2012).…”

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).…”