Integrated analysis of hsc70-based mammalian molecular chaperone system

哺乳动物胞质hsc70分子伴侣系统需要I型Dna J辅分子伴侣(S)参与线粒体蛋白的输入和变性蛋白的体外复性。我们报道了两个I型Dna J辅伴酮，DjA1和DjA2，广泛存在于哺乳动物组织中，在体外检测中功能相同。为了探讨DjA1和DjA2在小鼠中的功能，我们通过靶向干扰的方法获得了Dna A1缺失和Dna A2缺失的小鼠。小鼠Tom34基因有两个可供选择的起始外显子，转录的两个mRNAs仅在对应于两个起始外显子(外显子1a和1b)的5‘端序列上有所不同。带外显子1a的Tom34m RNA(Tom34a)普遍表达，而带外显子1b(Tom34b)的Tom34m RNA仅在成熟的睾丸生殖细胞中表达。Tom34-/-小鼠存活，生长正常。雄性Tom34-/-小鼠和雌性Tom34-/-小鼠都能生育。在体外将前蛋白导入分离的线粒体中，Tom34-/-小鼠和野生型小鼠之间没有明显差异。这些结果表明，在最佳条件下，Tom34对小鼠的生长发育是必不可少的。Dna jA1-/-和Dna jA2-/-雄性小鼠表现出轻微的生长迟缓，几乎是不育的。与DnajA1+/-和野生型小鼠相比，DnajA1+/-小鼠的睾丸重量减少了约50%。而来自DnajA2-/-小鼠的基因表达减少得更严重。DNAJA1-/-小鼠睾丸横切面显示粗线期精母细胞圆形细胞脱落，细胞凋亡率明显增加。RT-PCR分析显示，生殖细胞的几个阶段特异性基因的表达明显下降。然而，将GFP-精原细胞移植到DNajA1-/-小鼠体内，显示出支持体细胞的缺陷。而DnajA2-/-小鼠则存在精母细胞缺陷。

The mammalian cytosolic hsc70 chaperone system requires type I DnaJ cochaperone(s) for mitochondrial protein import and refolding of denatured proteins in vitro. We reported that two of type I DnaJ cochaperones, DjA1 and DjA2, are ubiquitously present in mammalian tissues and functionally equivalent in the in vitro assays. To explore the function of DjA1 and DjA2 in mouse, we generated DnajA1-null and DnajA2-null mice by targeted disruption. We also generated Tom34-null mice.The mouse Tom34 gene has two alternative initial exons and are transcribed two mRNAs that differs only in the 5'-proximal sequences corresponding to the two initial exons (exon 1a and 1b). Tom34 mRNA with exon 1a (Tom34a) is expressed ubiquitously, while that with exon 1b (Tom34b) is expressed only in mature testicular germ cells. The Tom34-/- mice were viable and grew normally. Male as well as female Tom34-/- mice were fertile. In vitro-preprotein import into isolated mitochondria showed no apparent difference between Tom34-/- and wild-type mice. These results indicate that Tom34 is dispensable for mouse growth and development under optimal conditions.The DnajA1-/- as well as DnajA2-/- male mice showed slight growth retardation and were almost sterile. The weights of testis from DnajA1-/- mice were reduced to about 50% compared to that of DnajA1+/- and wild-type littermates. While those from DnajA2-/- mice were more severely reduced. The cross-section of testis from DnajA1-/- mice revealed sloughing of round spermatids and apparent increase of apoptosis in pachytene-stage spermatocytes. RT-PCR analysis showed marked decreases in expression of several stage-specific genes of germ cells. However, transplantation of GFP-spermatogonia into DnajA1-/- mice indicated defect in supporting somatic cells. While DnajA2-/- mice had defect in spermatocyte.