Cytosolic Glycosylation

University Of Florida, Gainesville FL

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Abstract

DESCRIPTION (provided by applicant): The long-term goal of the study is to determine how and why proteins are modified by complex 0-linked glycans in the eukaryotic cytoplasm. Our immediate focus is on a pentasaccharide attached to a hydroxyproline on Skp1, a subunit of the multiprotein SCF E3 ubiquitin ligase, in Dictvostelium. Skp1 is modified by an entirely novel set of enzymes separate from those in the secretory pathway. Nearly all Skp1 is normally glycosylated but mutant forms are poorly and heterogeneously modified. The non-glycosylated forms failed to concentrate in the nucleus, an effect that could be recapitulated by disturbing glycosylation mutationally or pharmacologically. Based on this and additional biochemical evidence for Skp 1-enzyme interactions, we hypothesize that the Skp1 modification pathway has chaperone/quality control activity that facilitates and monitors folding of Skp1 for entry into the SCF complex and ultimately the nucleus. This model has parallels with the N-linked glycan-dependent chaperone/quality control retention system of the rER. in the coming project period, we will continue the study of 4 of the Skp1 modification enzymes with the ultimate goal of using the new-found information to test certain predictions of the chaperone/quality control hypothesis. Dictyostelium contains three 4-prolyl hydroxylase-like genes that are predicted to reside in the cytoplasm. We hypothesize that one of these modifies Skp1 and that a mutant Skp1 is poorly hydroxylated in vivo because of chaperone-like activity of the prolyl hydroxylase as in the rER. GnT5 1 copurifies with the GlcNAcTase activity and appears to be homologous to the mucin-type polypeptide aGalNAcTases of the Golgi. We hypothesize that GnT5l modifies Skp1, but poorly modifies mutant Skp1 in vivo because it forms a long-lived, catalysis-independent complex with it. The Bl,3GalTase and the al,2FucTase activities reside in the same protein and we will examine the hypothesis that it has a processive action to ensure rapid extension of the Skp1 glycan after the GlcNAc addition commitment step. We hypothesize that a partially-purified aGalTase activity adds one of the outer terminal a-Gal residues on Skp1, that it physically associates with an undergalactosylated precursor of the main pool of Skp1 until the Skp1 is ready to exit, and that mutant Skp1 will exhibit excessive recycling of its terminal Gal. These studies are expected to firmly identify the enzymes that modify Skp1 (except possibly for one of the aGalTases) and test specific hypotheses with regard to the chaperone/quality control model. In addition, the enzyme sequences are expected to be useful for the identification of homologous genes, in the cytoplasm or Golgi, of other 'lower' organisms as their genome sequences are completed.