Yeast glycosylation mutants are sensitive to aminoglycosides NETA

Aminoglycosides are a therapeutically important class of antibiotics that inhibit bacterial protein synthesis and a number of viral and eukaryotic functions by blocking RNA-protein interactions. Vanadate-resistant Saccharomyces cerevisiae mutants with defects in Golgi-specific glycosylation processes exhibit growth sensitivity to hygromycin B, an aminoglycoside [Ballou, L., Hitzeman, R. A., Lewis, M. S. & Ballou, C. E. (1991) Proc. Nall. Acad. Sci. USA 88,3209-3212]. Here, evidence is presented that glycosylation is, in and of itself, a key factor mediating aminoglycoside sensitivity in yeast. Examination ofmutants with a wide range of glycosylation abnormalities reveals that all are sensitive to aminoglycosides. This effect is specific to aminoglycosides and not merely a consequence of increased permeability of the yeast mutants to drugs. Furthermore, inhibition of glycosylation in wild-type cells leads to a marked increase in their sensitivity to aminoglycosides. These results establish that a defect in glycosylation is sufficient to render yeast cells susceptible to these clinically important drugs. Further, they suggest that a molecule which prevents the uptake or mediates removal of aminoglycosides requires glycosylation for its activity. Perhaps more importantly, this finding on drug sensitivity provides the most powerful screen to date to identify mutants and thereby to isolate genes involved in all aspects of N-linked glycosylation. Aminoglycosides are known to interfere with RNA-protein interactions. These antibiotics inhibit bacterial translation by interacting with the 16S ribosomal RNA (1-3). In eukaryotes, aminoglycosides block splicing of group I introns by binding to a specific RNA motif (4, 5). Certain members of the aminoglycoside family selectively block the binding of the human immunodeficiency virus (HIV) Rev protein to its cognate RNA recognition element (6). Inhibition ofHIV replication by members of this family has been demonstrated (6), qualifying aminoglycosides as important antiviral agents that likely mediate their effect through sequence-specific RNA interactions. However, the efficacy of these drugs as therapeutic agents is limited because of poor intracellular uptake in eukaryotic cells. Yeast mutants that have defects in Golgi-specific glycoprotein processing are selectively enriched by growth resistance to sodium vanadate and sensitivity to the aminoglycoside hygromycin B (7). Five hygromycin-sensitive complementation groups that have Golgi-specific glycosylation defects have been identified previously. Three of these are allelic to the well-characterized mnn8, mnn9, andmnnlO mutations, and the remaining two are allelic to the previously unidentified mutations vrgl and vrg4 (7). All of these mutants are defective in glycoprotein modifications that affect the elongation of outerchain carbohydrates in the Golgi complex (8). It has been proposed that these gene products function in an energydependent, Golgi-specific function. To identify more mutants defective in Golgi-specific functions, additional vanadateresistant/hygromycin B-sensitive cells were isolated. (These muThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tants and their genetic analyses will be described elsewhere.) Surprisingly, two mutants were isolated that are defective in very early steps of glycosylation that take place in or before the endoplasmic reticulum (ER). The isolation of mutants with defects in early steps in the glycosylation pathway suggested that hygromycin B sensitivity is not due to defects in Golgi-specific functions. To understand how a molecule that binds to RNA impinges upon glycosylation within the secretory pathway, I examined the effect of aminoglycosides on the growth characteristics of yeast glycosylation mutants. In the present work, I report the results of experiments that demonstrate that aminoglycoside hypersensitivity is due, at least in part, to defects in glycosylation. These experiments demonstrate that hygromycin B sensitivity is a phenotype that is not restricted to mutants with Golgi-specific glycosylation defects, but rather is common to a large number of yeast mutants with defects in all aspects of glycosylation. Drug sensitivity, therefore, provides a powerful selection technique that can be used for the cloning of the wild-type genes defined by these mutations. MATERIALS AND METHODS Strains, Plasmids, and Media. The following Saccharomyces cerevisiae strains were used in this study: DBY747-3D2 (MATa rad52-8::TRPl trpl-289 leu2-3,112 his3-AJ ura3-52) (R. Rothstein, Columbia University, New York); PRY55 (MATa algl-l ura3-52) (9); PRY212 (MATa alg2-1 ura3-52) (10); PRY90 (MATa alg3-1 ade2-101 ura3-52) (10); PRY95 (MA Ta alg4-4 ura3-52), PRY98 (MATa alg5-1 ade2-101 ura3-52) (10) (P. Robbins, Massachusetts Institute of Technology, Cambridge); RSY4 (MATa secl8-1 glsl-l) (11), LB1OB (MATa mnnl), LB116A (MATa mnn2), and TH210D (MAT a mnnl mnn2 ura3-52) (Berkeley Genetics Stock Center); mnn8 (MATa mnn8gall CUPJ), mnn9 (MATa mnn9gall CUP1), and mnnlO (MATa mnnlO gall CUP1) (C. Ballou, University of California, Berkeley); MA9-D (MATa wbpl-l ade2-101 his3-A200 ura3-52 lys2-801) (12) (M. Aebi, Eidgenossiche Technische Hochschule, Zurich); and 5762-6.2 (MATa cyh2 ura3-1 trpl ade2-101) (N. Hollingsworth, State University of New York). NDY1.3, -5.3, -9.3, -1.4, -13.4, and -17.4 are spontaneous vanadate-resistant mutants derived from MCY1093 (MAT a his4-539lys2-801 ura3-52) or MCY1094 (MATa ade2-101 ura352). NDY1.3, -9.3, and -13.4 may be novel mutants in that these are unable to complement previously identified vanadateresistant glycosylation mutants, or, in the case of NDY13.4, any early glycosylation mutants. NDY5.3 is allelic to mnn9. NDY1.4 and -17.4 are allelic to m163, an oligosaccharyltransferase mutant isolated from the Hartwell collection of mutants that are temperature sensitive for growth (13). These are derived from A364 (MATa adel ade2 ural lys2 tyrl his7gall-1). Wild-type strains used in this study include RSY255 (MA Ta ura3-52 leu2-2111) (R. Schekman, University of California, Berkeley) and W303 la (MATa ade2.l ura3-1 his3-11 trpl-l leu2-3,112 canl-100), A364 (MATa adel ade2 ural lys2 tyrl his7 gall-i); and SEY6210 (MATa ura3-52 his3-A200 trpl-A901 lys2-801 leu2-3,112 suc2-A9) (S. Emr, University of California, San Diego); SS328 (MATa ade2-101 his3A200 ura3-52 lys2Abbreviation: ER, endoplasmic reticulum.