Laboratory Exercises Transcriptional and Posttranslational Regulation of a Membrane Nutrient Transporter* Received for publication, May 9, 2002 Boris U. Stambuk‡ From the Departamento de Bioquı´mica, Centro de Cie ˆ ncias Biolo ´ gicas, Universidade Federal de Santa Catarina, Floriano ´ polis, SC 88040 –900, Brazil A laboratory exercise illustrating the metabolic regulation of a plasma membrane transporter was devised. A simple colorimetric assay allows the determination of the yeast maltose permease activity under different conditions, and thus the induction of the transporter by maltose and its repression by glucose can be easily demonstrated. The laboratory exercise also shows that glucose regulates the expression of the transporter at the plasma membrane by promoting its inactivation and endocytosis. The intracellular trafficking of the transporter under these conditions can be further illustrated using a maltose permease tagged with the green fluorescent protein. Keywords: Endocytosis, GFP, inactivation, lysosomal degradation, metabolic regulation, maltose permease, Saccharomyces cerevisiae, trafficking. The plasma membrane is an essential barrier between the cell and the extracellular milieu. Membrane proteins facilitate the uptake of nutrients and ions, mediate the communication between the exterior and interior of the cell, and also facilitate the efflux of toxic substances. Not surprisingly transmembrane proteins account for 20– 30% of the genes present in the genome of several organ- isms, and membrane transporters are one of the largest families of such proteins [1, 2]. Controlling the repertory and activity of plasma mem- brane proteins is a critical part of how cells respond to fluctuating extracellular signals and changing nutrient availability. While transcriptional regulation is one facet of such control, there is a growing appreciation of the impor- tance of posttranslational events (activation or inhibition, down-regulation, protein turnover, or trafficking) that reg- ulate plasma membrane proteins at the surface of the cell [3]. Indeed, several diseases (depression, hypertension, nutrient malabsorption, glucose intolerance, and cancer) are a direct consequence of deficient expression and/or regulation of plasma membrane receptors, transporters, or channels at the plasma membrane [4 –9]. Although there are many useful examples of laboratory exercises illustrating the transcription-regulated expres- sion of proteins and/or enzymes (e.g. Refs. 10 and 11), examples of laboratory exercises that show the posttrans- lational control of a protein are scarce. We have developed a very simple laboratory class that illustrates the transcrip- tional and posttranslational mechanisms involved in the regulated expression of a plasma membrane nutrient transporter using the model organism Saccharomyces cer- evisiae. This microorganism was chosen because of its GRAS (“generally regarded as safe”) status, easy mainte- nance in laboratory conditions, and well characterized physiology as well as the abundance of methods and tools for its genetic analysis and manipulation. Fermentation of maltose by S. cerevisiae is a critical phase in the processes of brewing and breadmaking. Malt- ose utilization requires three genes (MALR, MALT, and MALS), coding for a regulatory protein, the maltose trans- porter, and an -glucosidase, respectively. In the presence of maltose the constitutively expressed activator protein MalRp binds to the MALS and MALT promoters, inducing the coordinated transcription of these genes [10, 12, 13]. As a consequence, the permease actively transports malt- ose across the cell membrane, and subsequently the cy- toplasmic -glucosidase hydrolyzes maltose into two units of glucose, which are then metabolized through the gly- colytic pathway. However, glucose is a preferred carbon source for S. cerevisiae, and this sugar has an important regulatory function in the carbohydrate metabolism of yeasts. When glucose is present in the medium the expression of glu- coneogenic and alternative sugar-utilizing genes, including MAL genes, is inhibited by a mechanism known as glucose repression. Analysis of glucose repression has revealed several proteins and enzymes required for the cellular re- sponse to this sugar, and both glucose transport and phosphorylation are crucial upstream steps in the glucose sensing/signaling pathway. Indeed, glucose repression is * This work was supported in part by financial support from teaching improvement programs of Centro Argentino-Brasileiro de Biotecnologia-Conselho Nacional de Desenvolvimento Cien- tı´fico e Tecnolo ´ gico (No. 48.0146/00-4) and Fundo de Apoio ao Ensino de Graduac ¸a ˜ o-Universidade Federal de Santa Catarina. ‡ To whom correspondence should be addressed. Tel.: 55-48- 331-9589; Fax: 55-48-331-9672; E-mail: bstambuk@mbox1. ufsc.br. © 2002 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Printed in U.S.A. Vol. 30, No. 6, pp. 388 –393, 2002 This paper is available on line at http://www.bambed.org 388