Intracellular trafficking and ubiquitination of the Schizosaccharomyces pombe amino acid permease Aat1p

Abstract

In Schizosaccharomyces pombe, neither intracellular sorting nor ubiquitination of amino acid permeases is well understood. In the present study, we show that intracellular sorting of the amino acid permease Aat1p in S. pombe depends on the presence of a nitrogen source in the growth medium. Under nitrogen-sufficient conditions, Aat1p appeared to be stably localized at the Golgi apparatus. In contrast, under nitrogen-insufficient conditions, Aat1p was sorted to the plasma membrane. Over time, plasma membrane-localized Aat1p was internalized and sorted to the lumen of the vacuole, where it was degraded. Sorting of Aat1p to the vacuolar lumen was dependent on the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular body. S. pombe has three genes (pub1 + , pub2 + and pub3 + ) that are homologous to the ubiquitin ligase RSP5. Under nitrogen-sufficient conditions, Aat1-GFP was missorted to the plasma membrane in pub1D cells and ubiquitinated Aat1p was not detected. These results suggest that Pub1p-mediated ubiquitination is required for retention of Aat1 at the Golgi under nitrogen-sufficient conditions. The Aat1p lysine mutant Aat1 K18, 26, 27 was completely missorted to the plasma membrane under nitrogen-rich conditions. Furthermore, Aat1 K4, 18R , Aat1 K4, 26, 27R and Aat1 K18, 26, 27K mutants were severely blocked in endocytosis.These results indicate that ubiquitination is an important determinant for localization and regulation of the Aat1p permease in S. pombe.

Fission yeast Any1, β-arrestin-like protein, is involved in TSC-Rheb signaling and the regulation of amino acid transporters

“…Most newly synthesized transporters are ubiquitylated by the Pub1-Any1 complex at the Golgi when extracellular nutrients are abundant. This ubiquitylation is believed to be necessary for their storage in the Golgi (Nakase et al, 2012) but not for degradation. We would therefore propose that the Pub1-Any1 complex is responsible for storage of the transporters in cells growing under nutrient-rich condition.…”

“…Following a shift to nitrogen-poor medium, Aat1 is transported from the Golgi apparatus to the plasma membrane (PM), followed by endocytosis and transport to the vacuolar lumen (Nakase et al, 2010). Recently, we found that the membrane trafficking of Aat1 was regulated by the ubiquitin ligase Pub1, a homolog of Rsp5 sc , in fission yeast (Nakase et al, 2012). Pub1 to regulates G2/M transition by ubiquitylating Cdc25 (Nefsky and Beach, 1996), which dephosphorylates the inhibitory phosphotyrosine of Cdc2 (Dunphy and Kumagai, 1991;Gautier et al, 1991;Gould and Nurse, 1989;Lundgren et al, 1991;Russell and Nurse, 1986) and cooperates with Flp1/ Clp1 phosphatase (Esteban et al, 2008).…”

“…While Aat1 is found in the Golgi of wildtype cells under nitrogen-rich conditions, it is localized to the PM in Dpub1 cells. In addition, Aat1 is ubiquitylated in a Pub1-dependent manner (Nakase et al, 2012). This ubiquitylation is believed to be necessary for their storage in the Golgi (Nakase et al, 2012) but not for degradation (supplementary material Fig.…”

CUE Domain-Containing Protein Vps901 Is Required for Vacuolar Protein Transport inSchizosaccharomyces pombe

“…Recently, we reported that Aat1p was ubiquitinated and that ubiquitination is an important determinant of the localization and regulation of the Aat1p in S. pombe. 40) Thus the CUE domain of Vps901p may bind ubiquitinated Aat1p for efficient endocytosis from the plasma membrane to the vacuole. Studies undertaken to confirm this hypothesis should provide a deeper understanding of the role of the CUE domain in fission yeast.…”

Functional Expression ofSchizosaccharomyces pombeVba2p in the Vacuolar Membrane ofSaccharomyces cerevisiae

“…Although the plasma membrane protein(s) responsible for quinidine uptake have not been identified, a general amino acid permease, Aat1p of S. pombe, 14) a homolog of S. cerevisiae Gap1p, might be a site of competition of lysine with quinidine. However, it is also possible that S. pombe Vba2p mediates the uptake of quinidine into the vacuole, like lysine, and enhances the penetration of the quinidine into cells.…”