Integrated analysis of hsc70-based mammalian molecular chaperone system

哺乳动物细胞质hsc70伴侣系统需要I型DnaJ伴侣来进行线粒体蛋白的导入和体外变性蛋白的再折叠。我们报道了两种I型DnaJ合作伴侣，DjA1和DjA2，普遍存在于哺乳动物组织中，并且在体外实验中功能相同。为了探索DjA1和DjA2在小鼠中的功能，我们通过靶向破坏产生了DnajA1-null和DnajA2-null小鼠。我们还生成了Tom34-null小鼠。小鼠Tom34基因有两个可选的初始外显子，转录的两个mrna仅在与两个初始外显子（1a和1b外显子）对应的5'-近端序列上不同。带有外显子1a （Tom34a）的Tom34 mRNA普遍表达，而带有外显子1b （Tom34b）的mRNA仅在成熟睾丸生殖细胞中表达。Tom34-/-小鼠存活，生长正常。雄性和雌性Tom34-/-小鼠均可生育。在离体实验中，Tom34-/-和野生型小鼠的线粒体前蛋白输入量没有明显差异。这些结果表明，在最佳条件下，Tom34对于小鼠的生长发育是必不可少的。DnajA1-/-和DnajA2-/-雄性小鼠表现出轻微的生长迟缓，几乎不育。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-/-的小鼠则更严重。DnajA1-/-小鼠睾丸横切面显示圆形精子细胞脱落，粗线期精子细胞凋亡明显增加。RT-PCR分析显示，生殖细胞中几个阶段特异性基因的表达明显降低。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-/-小鼠精母细胞存在缺陷。

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.