Internalization of Heterologous Sugar Transporters by Endogenous α-Arrestins in the Yeast Saccharomyces cerevisiae

Abstract

When expressed in Saccharomyces cerevisiae using either of two constitutive yeast promoters (PGK1prom and CCW12prom), the transporters CDT-1 and CDT-2 from the filamentous fungus Neurospora crassa are able to catalyze, respectively, active transport and facilitated diffusion of cellobiose (and, for CDT-2, also xylan and its derivatives). In S. cerevisiae, endogenous permeases are removed from the plasma membrane by clathrin-mediated endocytosis and are marked for internalization through ubiquitinylation catalyzed by Rsp5, a HECT class ubiquitin:protein ligase (E3). Recruitment of Rsp5 to specific targets is mediated by a 14-member family of endocytic adaptor proteins, termed α-arrestins. Here we demonstrate that CDT-1 and CDT-2 are subject to α-arrestin-mediated endocytosis, that four α-arrestins (Rod1, Rog3, Aly1, and Aly2) are primarily responsible for this internalization, that the presence of the transport substrate promotes transporter endocytosis, and that, at least for CDT-2, residues located in its C-terminal cytosolic domain are necessary for its efficient endocytosis. Both α-arrestin-deficient cells expressing CDT-2 and otherwise wild-type cells expressing CDT-2 mutants unresponsive to α-arrestin-driven internalization exhibit an increased level of plasma membrane-localized transporter compared to that of wild-type cells, and they grow, utilize the transport substrate, and generate ethanol anaerobically better than control cells.IMPORTANCE Ethanolic fermentation of the breakdown products of plant biomass by budding yeast Saccharomyces cerevisiae remains an attractive biofuel source. To achieve this end, genes for heterologous sugar transporters and the requisite enzyme(s) for subsequent metabolism have been successfully expressed in this yeast. For one of the heterologous transporters examined in this study, we found that the amount of this protein residing in the plasma membrane was the rate-limiting factor for utilization of the cognate carbon source (cellobiose) and its conversion to ethanol.

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillusoryzae

“…In fact, the yeast maltose transporter Mal21, which is resistant to glucose-induced endocytosis, showed higher maltose uptake activity than that of Mal61 (31). In addition, deletion of four arrestin-like proteins, including Rod1, increased the cellobiose fermentation of yeast expressing cellobiose transporters from the filamentous fungus Neurospora crassa (32). However, creD disruption had a negative effect on ␣-amylase production, whereas MalP was still retained at the plasma membrane in the presence of glucose (Fig.…”

Studies on sugar transporter CRT1 reveal new characteristics that are critical for cellulase induction in Trichoderma reesei

“…The authors also discovered that the truncation of the C-terminal tail of STP1 by 16 amino acids improved growth significantly on glucose, while no growth was seen on glucose if the truncation was extended to 32 amino acids. The intracellular parts have also been shown to be involved in substrate-induced ubiquitination and subsequent degradation of N. crassa cellodextrin transporters when heterologously expressed in yeast [ 30 ]. Analysis of the CRT1 amino acid sequence by UbPred ubiquitination site prediction server [ 31 ] gave two possible ubiquitination sites, both located in the N-terminal intracellular part of the protein.…”

Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

“…Although both ST2 and ST16 did not improve anaerobic utilization of XD compared with CDT-2, the presence of xylose or other simple sugars could be used to initiate the consumption of XD, according to our previous results [ 7 ]. Based on the rapid turn-off and turn-on of XD consumption in anaerobic conditions [ 7 ], we suspect that the XD transporters are internalized in the absence of hexose or xylose sugars [ 27 ]. Currently, how CDT-2 and ST16 are regulated in xylodextrin-only media remains unknown.…”