Bredeston Luis M, Marino-Buslje Cristina, Mattera Vanesa S, Buzzi Lucila I, Parodi Armando J, D'Alessio Cecilia
Department of Biological Chemistry and IQUIFIB (CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires C1113AAD, Argentine.
Fundación Instituto Leloir and IIBBA, CONICET. Av. Patricias Argentinas 435, Buenos Aires C1405BWE, Argentine.
Glycobiology. 2017 Jan;27(1):64-79. doi: 10.1093/glycob/cww092. Epub 2016 Sep 1.
UDP-Glc entrance into the endoplasmic reticulum (ER) of eukaryotic cells is a key step in the quality control of glycoprotein folding, a mechanism requiring transfer of a Glc residue from the nucleotide sugar (NS) to glycoprotein folding intermediates by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). According to a bioinformatics search there are only eight genes in the Schizosaccharomyces pombe genome belonging to the three Pfam families to which all known nucleotide-sugar transporters (NSTs) of the secretory pathway belong. The protein products of two of them (hut1 and yea4) localize to the ER, those of genes gms1, vrg4, pet1, pet2 and pet3 to the Golgi, whereas that of gms2 has an unknown location. Here we demonstrate that (1) Δhut1 and Δgpt1 (UGGT null) mutants share several phenotypic features; (2) Δhut1 mutants show a 50% reduction in UDP-Glc transport into ER-derived membranes; (3) in vivo UDP-Glc ER entrance occurred in Δhut1Δyea4Δgms2 mutants and in cells in which Δhut1 disruption was combined with that of each of four of the genes encoding Golgi-located proteins. Therefore, disruption of all genes whose products localize to the ER or have an unknown location did not obliterate UDP-Glc ER entrance. We conclude that the hut1 gene product is involved in UDP-Glc entrance into the ER, but that at least another as yet unknown NST displaying an unconventional sequence operates in the yeast secretory pathway. This conclusion agrees with our previous results showing that UDP-Glc entrance into the yeast ER does not follow the classical NST antiport mechanism.
UDP-葡萄糖进入真核细胞的内质网（ER）是糖蛋白折叠质量控制的关键步骤，这一机制需要UDP-葡萄糖：糖蛋白葡萄糖基转移酶（UGGT）将葡萄糖残基从核苷酸糖（NS）转移到糖蛋白折叠中间体上。根据生物信息学搜索，裂殖酵母基因组中只有八个基因属于分泌途径中所有已知核苷酸糖转运蛋白（NST）所属的三个Pfam家族。其中两个基因（hut1和yea4）的蛋白质产物定位于内质网，gms1、vrg4、pet1、pet2和pet3基因的产物定位于高尔基体，而gms2基因的产物定位未知。在这里，我们证明：（1）Δhut1和Δgpt1（UGGT缺失）突变体具有几个共同的表型特征；（2）Δhut1突变体中UDP-葡萄糖向内质网衍生膜的转运减少了50%；（3）在Δhut1Δyea4Δgms2突变体以及Δhut1缺失与四个编码高尔基体定位蛋白的基因中的每一个基因缺失相结合的细胞中，发生了体内UDP-葡萄糖进入内质网的过程。因此，破坏所有其产物定位于内质网或定位未知的基因并没有消除UDP-葡萄糖进入内质网的过程。我们得出结论，hut1基因产物参与UDP-葡萄糖进入内质网的过程，但至少还有另一种具有非常规序列的未知NST在酵母分泌途径中起作用。这一结论与我们之前的结果一致，即UDP-葡萄糖进入酵母内质网并不遵循经典的NST反向转运机制。
