Consequences of Disrupting the Gene That Encodes -Glucosidase II in the N-linked Oligosaccharide Biosynthesis Pathway of Dictyostelium discoideum HUDSON H. FREEZE, 1 * MARION LAMMERTZ, 1,3 NEGIN IRANFAR, 2 DANNY FULLER, 2 K. PANNEERSELVAM, 1 AND WILLIAM F. LOOMIS 2 1 The Burnham Institute, La Jolla, California 2 Department of Biology, University of California San Diego, La Jolla, California 3 Bergische Universitat-GH Wuppertal, Department of Biochemie, Wuppertal, Germany ABSTRACT W e have identified and disrupted the gene coding for -glucosidase II in Dictyostelium discoideum. This enzyme is responsible for removing two  1,3-linked glucose residues from N -linked oligosaccha- rides on newly synthesized glycoproteins. M utagenesis by restriction enzyme-mediated integration (REM I) gener- ated a clone, DG1033, which grows well but forms abnormal fruiting bodies with short, thick stalks. The strain lacks -glucosidase II activity and makes incom- pletely processed N -linked oligosaccharides that are abnormally large and have fewer sulfate and phosphate esters. The morphological, enzymatic, and oligosaccha- ride profile phenotypes of the disruption mutant are all recapitulated by a targeted disruption of the normal gene. Furthermore, all of these defects are corrected in cells transformed with a normal, full-length copy of the gene. The phenotypic characteristics of DG1033 as well as chromosomal mapping of the disrupted gene indicate that it is the site of the previously characterized modA mutation. The Dictyostelium gene ishighly homolo- gous to -glucosidase II genes in the human and the pig, C. elegans , and yeast. Although various cell lines have been reported to be defective in -glucosidase II activity, disruption of the Dictyostelium gene gives the first ex- ample of a clear developmental phenotype associated with loss of this enzyme. Dev. Genet. 21:177–186, 1997.  1 9 9 7 W iley-Liss, Inc. Key words: N -linked oligosaccharide; Dictyostelium; -glucosidase; development INTRODUCTION Many proteins trafficking through the eukaryotic ER and Golgi contain N-linked oligosaccharide chains that are assembled on a dolichol carrier in the ER and co-translationally added to newly synthesized proteins [Kornfeld and Kornfeld, 1985]. The sugar chain is composed of 2 N-acetylglucosamine, 9 mannose, and 3 tandem glucose residues located on one of the anten- nae. Shortly after transfer to protein, the glucose residues are removed by a set of two ER-localized neutral -glucosidases. The first enzyme, -glucosidase I, removes the terminal 1,2 glucose residue and the second enzyme, -glucosidase II, is responsible for removing the next two 1,3 glucose units. In mamma- lian cells, the oligosaccharides are often further trimmed by several neutral -mannosidases in the Golgi appara- tus before being converted into complex chains contain- ing additional N-acetylglucosamine, galactose and si- alic acids [Kornfeld and Kornfeld, 1985]. The soil amoebae Dictyostelium discoideum also syn- thesizes typical glucosylated sugar chains and degluco- sylates them, using a pair of glucosidases similar to those of other eukaryotes [Freeze, 1991]. It is unlikely that Dictyostelium makes complex type N-linked chains similar to those in mammalian cells (Ivatt et al., 1984; Amatayakul-Chantler et al., 1991; Freeze, 1991), al- though a Dictyostelium cell adhesion protein is suggested to have complex-type chains (Yoshida et al., 1993). There is very little cleavage of mannose from oligosac- charides of growing Dictyostelium cells [Sharkey and Kornfeld, 1991a,b; Freeze, 1991]; however, some sugar chains, especially those on lysosomal enzymes, are phosphorylated and sulfated to produce highly charged oligosaccharides [Freeze et al., 1990; Cardelli et al., 1990a; Freeze, 1991; Cardelli, 1993]. These chains often contain an ‘‘intersecting’’ -N-acetylglucosamine resi- due [Freeze et al., 1983b; Couso et al., 1987; Sharkey and Kornfeld, 1989, 1991a]. Some neutral chains have a -fucose residue in the chitobiose core [Ivatt et al., Contract grant sponsor: GM32485 (to H.H.F.); Contract grant sponsor: HD30892 (to W.F.L.). Marion Lammertz is currently at Bergische Universitat-GH Wupper- tal, Department of Biochemie, Wuppertal, Germany. *Correspondence to: Hudson H. Freeze, The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Received 23 June 1997; accepted 13 August 1997. DEVELOPMENTAL GENETICS 21:177–186 (1997)  1997 WILEY-LISS, INC.