Cytosolic Glycosylation

描述（由申请人提供）：研究的长期目标是 确定蛋白质如何以及为什么被复杂的0-连接聚糖修饰， 真核细胞质我们当前的焦点是一种五糖， Skp 1上的羟脯氨酸，Skp 1是多蛋白SCF E3泛素连接酶的亚基， 在独裁统治中。Skp 1被一组全新的酶修饰， 与分泌途径中的不同几乎所有Skp 1都是正常糖基化的 但突变体形式的修饰较差且不均匀。非糖基化 形式未能集中在细胞核中，这可能是一种影响， 通过突变或突变干扰糖基化来概括。 基于这一点以及Skp 1酶的其他生化证据， 相互作用，我们假设Skp 1修饰途径 伴侣/质量控制活动，促进和监测折叠 Skp 1进入SCF复合体并最终进入细胞核。该模型具有 与N-连接聚糖依赖性伴侣/质量控制平行 rER的保留系统。在未来的计划期间，我们会继续 研究4种Skp 1修饰酶，最终目标是使用 新发现的信息，以测试某些预测的伴侣/质量 控制假说Dictyosteroid含有三个4-脯氨酰羟化酶样 被预测存在于细胞质中的基因。我们假设 这些修饰了Skp 1，突变体Skp 1在体内羟基化较差 因为在rER中脯氨酰羟化酶的伴侣样活性。 GnT 5 1与GlcNAcTase活性共纯化，并且似乎与GlcNAcTase同源。 高尔基体的粘蛋白型多肽α GalNAcTases。我们假设GnT 5l 修饰Skp 1，但在体内对突变体Skp 1的修饰很差，因为它形成了一个 B1，3GalTase和B1，3GalTase与B1，3GalTase形成了一种长寿命的、不依赖催化的复合物。 al，2FucTase活性存在于相同的蛋白质中，我们将检查 假设它有一个进行性的行动，以确保快速扩展的 GlcNAc添加承诺步骤后的Skp 1聚糖。我们假设 部分纯化的aGalTase活性添加了外末端a-Gal之一， Skp 1上的残基，它与半乳糖基化不足的 Skp 1的主池的前体，直到Skp 1准备好退出，并且 突变体Skp 1将表现出其末端Gal的过度再循环。这些研究 预计可以确定修饰Skp 1的酶（除了可能的 对于aGalTase之一），并测试关于aGalTase的特定假设。 伴侣/质量控制模型。此外，预计酶序列 可用于鉴定细胞质中的同源基因，或 高尔基，其他'较低'的有机体，因为他们的基因组序列完成。

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.