title: Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.
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 β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-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 identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America.
authors.forename: Wenhan
authors.initials: W
authors.lastname: Zhu
authors.forename: Simran
authors.initials: S
authors.lastname: Banga
authors.forename: Yunhao
authors.initials: Y
authors.lastname: Tan
authors.forename: Cheng
authors.initials: C
authors.lastname: Zheng
authors.forename: Robert
authors.initials: R
authors.lastname: Stephenson
authors.forename: Jonathan
authors.initials: J
authors.lastname: Gately
authors.forename: Zhao-Qing
authors.initials: ZQ
authors.lastname: Luo
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D016952:Legionella pneumophila / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D019151:Peptide Library; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases