Alteration of haem‐attachment and signal‐cleavage sites for Paracoccus denitrificans cytochrome c550 probes pathway of c‐type cytochrome biogenesis in Escherichia coli
Alteration of haem‐attachment and signal‐cleavage sites for Paracoccus denitrificans cytochrome c550 probes pathway of c‐type cytochrome biogenesis in Escherichia coli
复制标题
脱氮副球菌细胞色素 c550 血红素附着和信号裂解位点的改变探针大肠杆菌 c 型细胞色素生物发生途径
作者：
Y. Sambongi;R. Stoll;S. Ferguson
脱氮副球藻细胞色素C550在大肠杆菌中以周质全蛋白的形式表达，血结合基序(Cys-X-X-Cys-His)中半胱氨酸残基的氨基酸替换共同或单独地阻止了血红素的共价结合，但不能阻止多肽移位到周质中。当天然信号裂解位点-3至-1位的3个丙氨酸残基被删除，或-1位的丙氨酸变为谷氨酰胺时，信号裂解发生在交替位置(后者仅有10个残基)，但仍发生血红素附着。当同样的三种丙氨酸被改变为天冬氨酸-谷氨酸-天冬氨酸时，检测到保留了完整信号序列的膜相关载脂蛋白产物。这些和其他结果表明：(I)血红素附着不是脱细胞色素C550出口到周质所必需的；(Ii)血红素当附着在细胞膜上时不能附着到细胞色素C550上，这表明信号序列切割先于周质血红素附着，这可能发生在离成熟的N-末端只有六个残基的地方；(Iii)血红素附着需要两个半胱氨酸，可能是因为二硫键是中间体。酿酒酵母线粒体iso-1-细胞色素c基因与成熟细胞色素C550的信号序列和前10个残基融合后，在大肠杆菌中以全蛋白的形式表达，表明该基因具有广泛的底物特异性。
Paracoccus denitrificans cytochrome c550 is expressed as a periplasmic holo‐protein in Escherichia coli; amino acid substitutions of cysteine residues in the haembinding motif (Cys‐X‐X‐Cys‐His), either together or singly, prevented covalent attachment of haem but not polypeptide translocation into the periplasm. When the three alanine residues at positions ‐3 to ‐1 in the native signal‐cleavage site were deleted, or alanine at ‐1 was changed to glutamine, signal cleavage was at alternative sites (after only ten residues in the latter case), but haem attachment still occurred. When the same three alanines were changed to Asp‐Glu‐Asp, a membrane‐associated apo product that had retained the complete signal sequence was detected. These and other results presented here indicate that (i) haem attachment is not required for the apo‐cytochrome c550 export to the periplasm; (ii) haem cannot attach to apocytochrome c550 when attached to the cytoplasmic membrane, suggesting that signal‐sequence cleavage precedes periplasmic haem attachment, which can occur at as few as six residues from the mature N‐terminus; and (iii) two cysteines are required for haem attachment, possibly because a disulphide bond is an intermediate. The gene for Saccharomyces cerevisiae mitochondrial iso‐1‐cytochrome c was expressed as a holo‐protein in E. coli when fused with the signal sequence plus the first 10 residues of the mature cytochrome c550, indicating that the E. coli cellular apparatus for the c‐type cytochrome biogenesis has a broad substrate specificity.
DOI：
10.1016/s0021-9258(19)84657-4
发表时间：
1989-10
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
M. Drygas;A. Lambowitz;F. Nargang
通讯作者：
M. Drygas;A. Lambowitz;F. Nargang
影响因子：
11.4
作者：
DUMONT, ME;ERNST, JF;SHERMAN, F
通讯作者：
SHERMAN, F