Sommer J M, Nguyen T T, Wang C C
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
FEBS Lett. 1994 Aug 15;350(1):125-9. doi: 10.1016/0014-5793(94)00747-0.
Import of proteins into the glycosomes of T. brucei resembles the peroxisomal protein import in that C-terminal SKL-like tripeptide sequences can function as targeting signals. Many of the glycosomal proteins do not, however, possess such C-terminal tripeptide signals. Among these, phosphoenolpyruvate carboxykinase (PEPCK (ATP)) was thought to be targeted to the glycosomes by an N-terminal or an internal targeting signal. A limited similarity to the N-terminal targeting signal of rat peroxisomal thiolase exists at the N-terminus of T. brucei PEPCK. However, we found that this peroxisomal targeting signal does not function for glycosomal protein import in T. brucei. Further studies of the PEPCK gene revealed that the C-terminus of the predicted protein does not correspond to the previously deduced protein sequence of 472 amino acids due to a -1 frame shift error in the original DNA sequence. Readjusting the reading frame of the sequence results in a predicted protein of 525 amino acids in length ending in a tripeptide serine-arginine-leucine (SRL), which is a potential targeting signal for import into the glycosomes. A fusion protein of firefly luciferase, without its own C-terminal SKL targeting signal, and T. brucei PEPCK is efficiently imported into the glycosomes when expressed in procyclic trypanosomes. Deletion of the C-terminal SRL tripeptide or the last 29 amino acids of PEPCK reduced the import only by about 50%, while a deletion of the last 47 amino acids completely abolished the import. These results suggest that T. brucei PEPCK may contain a second, internal glycosomal targeting signal upstream of the C-terminal SRL sequence.
蛋白质导入布氏锥虫的糖体过程类似于过氧化物酶体蛋白质导入，因为C端类似SKL的三肽序列可作为靶向信号。然而，许多糖体蛋白并不具备此类C端三肽信号。其中，磷酸烯醇式丙酮酸羧激酶（PEPCK (ATP)）被认为是通过N端或内部靶向信号靶向至糖体的。布氏锥虫PEPCK的N端与大鼠过氧化物酶体硫解酶的N端靶向信号存在有限的相似性。然而，我们发现这种过氧化物酶体靶向信号在布氏锥虫的糖体蛋白导入过程中不起作用。对PEPCK基因的进一步研究表明，由于原始DNA序列中的-1移码错误，预测蛋白质的C端与先前推导的472个氨基酸的蛋白质序列不对应。调整序列的阅读框后，得到一个预测长度为525个氨基酸的蛋白质，其末端为三肽丝氨酸-精氨酸-亮氨酸（SRL），这是一个潜在的导入糖体的靶向信号。萤火虫荧光素酶的融合蛋白（无自身C端SKL靶向信号）与布氏锥虫PEPCK在原循环锥虫中表达时可有效导入糖体。删除C端SRL三肽或PEPCK的最后29个氨基酸仅使导入减少约50%，而删除最后47个氨基酸则完全消除了导入。这些结果表明，布氏锥虫PEPCK可能在C端SRL序列上游包含第二个内部糖体靶向信号。
