This application is concerned with glycosylation and glycoprotein processing. The relevant enzymes are localized in the endoplasmic reticulum (ER) and Golgi. In the glycosylation area, recent emphasis in our laboratory has been on basic questions of topography, organization, and mechanism of action of individual enzymes. Specific aims for the coming grant period include continued study of the topography of dolichol phosphate mannose synthase, the cloning and sequencing of dolichol phosphate glucose synthase, and further exploration of a 13 amino acid peptide sequence in ER glycosyltransferases that may serve as a recognition site for the polyisoprenoid compound, dolichol phosphate. Other ER projects include isolation and sequencing of Dol-P-Man synthase cDNAs from mammalian tissues and the cloning of the yeast ALG3 gene, a gene that may encode an enzyme which catalyzes transmembrane movement of dolichol pyrophosphate linked oligosaccharides. In parallel with this program in glycosylation, a program concerning basic mechanisms of glycoprotein is being developed. With the help of yeast molecular biology, current models for nucleotide sugar transport from the cytoplasm into Golgi vesicles are being tested. We are also beginning to clone and characterize yeast Golgi mannosyl transferases. The cloning and sequencing of mammalian Golgi processing mannosidases represents another major emphasis. The sequencing of mannosidase II has recently been completed and in collaboration with Dr. John Schutzbach we have extended this program to the cloning of liver alpha1,2-mannosidase I. Attempts will be made to express both mammalian mannosidases in yeast to determine whether functional Golgi localization signals are shared between yeast and mammals. Finally, in collaboration with Dr. Michiko Fukuda we will investigate a case of the genetic disease HEMPAS. Preliminary data indicate that this case may be associated with lower than normal levels of mannosidase II.