comprehensive identification of protein substrates of the doticm type iv transporter of legionella pneumophila综合识别的蛋白质基质嗜肺性军团菌的doticm iv型运输机.pdfVIP

Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila 1 1. 1. 2 1 1 Wenhan Zhu , Simran Banga , Yunhao Tan , Cheng Zheng , Robert Stephenson , Jonathan Gately , Zhao-Qing Luo1* 1 Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America, 2 Department of Statistics, Purdue University, West Lafayette, Indiana, United States of America Abstract A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of b-lactamase. The transfer of the fusions into mammalian cells was determined using the b-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have ide