Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the 1d1Cp complex
Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the 1d1Cp complex
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DOI：
10.1073/pnas.96.3.915
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发表时间：
1999-02-02
影响因子：
11.1
通讯作者：
Krieger, M
中科院分区：
文献类型：
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作者：
Chatterton, JE;Hirsch, D;Krieger, M
中国仓鼠卵巢（CHO）细胞突变体ldlC和ldlB，表现出几乎相同的表型，定义了两个基因所需的多个步骤，在正常的中间和transGolgi相关的处理的糖缀合物。LDLC基因编码ldlCp，一种约80-kDa的蛋白质，其在野生型而非ldlB细胞中可逆地与高尔基体的细胞质表面缔合。在这里，我们已经使用了逆转录病毒为基础的表达克隆系统来克隆小鼠的cDNA，LDLB，纠正多效性突变的ldlB细胞的表型。在ldlB突变体中未检测到相应的mRNA。LDLB编码一个约110-kDa的蛋白，ldlBp，它缺乏同源性已知的蛋白质，不包含共同的结构基序。数据库搜索确定了短片段的同源性从果蝇，拟南芥，和秀丽隐杆线虫的序列，和基本上全长同源的人类序列（82%的同一性），但是，是ldlCp的情况下，没有同源物被确定在酿酒酵母。我们已经发现，在野生型细胞胞质溶胶中，ldlCp是约950-kDa的“ldlCp复合物”的组分，其在ldlB胞质溶胶中较小，约为700 kDa。该复合物的正常组装是ldlBp依赖性的，并且可能是ldlCp的高尔基体缔合和多个腔高尔基体过程的正常活动所需的。
The Chinese hamster ovary (CHO) cell mutants ldlC and ldlB, which exhibit almost identical phenotypes, define two genes required for multiple steps in the normal medial and trans Golgi-associated processing of glycoconjugates. The LDLC gene encodes ldlCp, an approximate to 80-kDa protein, which in wild-type, but not ldlB, cells associates reversibly with the cytoplasmic surface of the Golgi apparatus. Here, we have used a retrovirus-based expression cloning system to clone a murine cDNA, LDLB, that corrects the pleiotropic mutant phenotypes of ldlB cells. The corresponding mRNA was not detected in ldlB mutants. LDLB encodes an approximate to 110-kDa protein, ldlBp, which lacks homology to known proteins and contains no common structural motifs. Database searches identified short segments of homology to sequences from Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans, and the essentially fall-length homologous human sequence (82% identity); however, as was the case for ldlCp, no homologue was identified in Saccharomyces cerevisiae. We have found that in wild-type cell cytosols, ldlCp is a component of an approximate to 950-kDa "ldlCp complex," which is smaller, approximate to 700 kDa, in ldlB cytosols. Normal assembly of this complex is ldlBp-dependent and may be required for Golgi association of ldlCp and for the normal activities of multiple luminal Golgi processes.