Internalization Of Heterologous Cellodextrin Transporters Is Mediated By Endogenous Alpha-Arrestins In Saccharomyces Cerevisiae

crossmark Internalization of Heterologous Sugar Transporters by Endogenous !-Arrestins in the Yeast Saccharomyces cerevisiae Arpita Sen, a,c Ligia Acosta-Sampson, a,c Christopher G. Alvaro, a Jonathan S. Ahn, a,d Jamie H. D. Cate, a,b,c Jeremy Thorner a,c ABSTRACT When expressed in Saccharomyces cerevisiae using either of two constitutive yeast promoters (PGK1 prom and CCW12 prom ), the transporters CDT-1 and CDT-2 from the filamentous fungus Neurospora crassa are able to catalyze, respectively, active trans- port and facilitated diffusion of cellobiose (and, for CDT-2, also xylan and its derivatives). In S. cerevisiae, endogenous per- meases 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 medi- ated by a 14-member family of endocytic adaptor proteins, termed !-arrestins. Here we demonstrate that CDT-1 and CDT-2 are subject to !-arrestin-mediated endocytosis, that four !-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, resi- dues located in its C-terminal cytosolic domain are necessary for its efficient endocytosis. Both !-arrestin-deficient cells express- ing CDT-2 and otherwise wild-type cells expressing CDT-2 mutants unresponsive to !-arrestin-driven internalization exhibit an increased level of plasma membrane-localized transporter compared to that of wild-type cells, and they grow, utilize the trans- port 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 at- tractive biofuel source. To achieve this end, genes for heterologous sugar transporters and the requisite enzyme(s) for subse- quent 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. E thanol is a widely used, environmentally clean, and renewable biofuel produced by microbial fermentation of sugar sources derived from food-related crop plants, such as corn and sugar cane, referred to as “first-generation” ethanol (1, 2). An alterna- tive source of ethanol that avoids the “food versus fuel” ethical conflict is sugar derived from non-crop plant biomass, referred to as “second-generation” ethanol (2, 3). Plant biomass is composed of lignocellulosic material, which consists of cellulose (the most abundant fraction), hemicellulose, and lignin (4). For its fermen- tation to occur, lignocellulosic biomass is first pretreated to make its components more accessible to breakdown and then hydro- lyzed either enzymatically or chemically to release fermentable sugars (5). The principal sugars liberated by hydrolysis of cellulose consist of cellodextrins and glucose, whereas hydrolysis of hemi- celluloses releases primarily xylans and xylose. To produce ethanol as a biofuel, industrial strains of budding yeast Saccharomyces cerevisiae are primarily used (6, 7). Native S. cerevisiae, although unable to efficiently utilize xylose (8, 9), is proficient in the utilization and fermentation of glucose. How- ever, large-scale enzymatic degradation of cellulose into glucose is expensive, requiring, first, hydrolysis of cellulose by cellulases to generate the (1i4)-linked disaccharide cellobiose (and higher cellodextrins) and then subsequent cleavage of cellobiose into glu- cose by -glucosidases. Aside from the expense, complete enzy- matic conversion of cellulose to glucose is problematic because high glucose concentrations inhibit both cellulases and -gluco- sidases (10, 11). One approach that reduces cost, eliminates glu- aem.asm.org cose-mediated inhibition of enzymes, and facilitates cofermenta- tion of nonglucose sugars is based on the successful uptake of cellobiose, which is subsequently broken down to glucose after its transport into the cell. This end was achieved by ectopic coexpres- sion in yeast of the gene for a cellobiose/cellodextrin transporter, either CDT-1 (NCU00801) or CDT-2 (NCU08114), and the gene for an intracellular -glucosidase (gh1-1, NCU00130) from the filamentous fungus Neurospora crassa (12). CDT-1 catalyzes ac- tive transport of cellobiose, and CDT-2 mediates entry of cellobi- ose (as well as xylans) by facilitated diffusion (12, 13). Cellobiose fermentation, like fermentation of other nonglucose sugars in S. cerevisiae, occurs at a substantially lower rate than glucose fermen- Received 20 July 2016 Accepted 23 September 2016 Accepted manuscript posted online 30 September 2016 Citation Sen A, Acosta-Sampson L, Alvaro CG, Ahn JS, Cate JHD, Thorner J. 2016. Internalization of heterologous sugar transporters by endogenous !-arrestins in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 82:7074 –7085. doi:10.1128/AEM.02148-16. Editor: M. J. Pettinari, University of Buenos Aires Address correspondence to Jeremy Thorner, jthorner@berkeley.edu. A.S. and L.A.-S. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.02148-16. Copyright © 2016 Sen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Applied and Environmental Microbiology December 2016 Volume 82 Number 24 Downloaded from http://aem.asm.org/ on March 28, 2017 by University of California, Berkeley Department of Molecular and Cell Biology, a Department of Chemistry, b Energy Biosciences Institute, c and School of Public Health, d University of California, Berkeley, California, USA