Functional dissection of the XpsN (GspC) protein of the Xanthomonas campestris pv. campestris type II secretion machinery.
Type II secretion machinery is composed of 12 to 15 proteins for translocating extracellular proteins across the outer membrane. XpsL, XpsM, and XpsN are components of such machinery in the plant pathogen Xanthomonas campestris pv. campestris. All are bitopic cytoplasmic-membrane proteins, each with a large C-terminal periplasmic domain. They have been demonstrated to form a dissociable ternary complex. By analyzing the C-terminally truncated XpsN and PhoA fusions, we discovered that truncation of the C-terminal 103 residues produced a functional protein, albeit present below detectable levels. Furthermore, just the first 46 residues, encompassing the membrane-spanning sequence (residues 10 to 32), are sufficient to keep XpsL and XpsM at normal abundance. XpsN46(His6), synthesized in Escherichia coli, is able to associate in a membrane-mixing experiment with the XpsL-XpsM complex preassembled in X. campestris pv. campestris. The XpsN N-terminal 46 residues are apparently sufficient not only for maintaining XpsL and XpsM at normal levels but also for their stable association. The membrane-spanning sequence of XpsN was not replaceable by that of TetA. However, coimmunoprecipitation with XpsL and XpsM was observed for XpsN97::PhoA, but not XpsN46::PhoA. Only XpsN97::PhoA is dominant negative. Single alanine substitutions for three charged residues within the region between residues 47 and 97 made the protein nonfunctional. In addition, the R78A mutant XpsN protein was pulled down by XpsL-XpsM(His6) immobilized on an Ni-nitrilotriacetic acid column to a lesser extent than the wild-type XpsN. Therefore, in addition to the N-terminal 46 residues, the region between residues 47 and 97 of XpsN probably also plays an important role in interaction with XpsL-XpsM.
Lee
Hsien-Min
HM
Institute of Biochemistry, National Chung Hsing University, Taichung, Taiwan, Republic of China.
Chen
Juine-Ruey
JR
Lee
Hai-Lun
HL
Leu
Wei-Ming
WM
Chen
Ling-Yun
LY
Hu
Nien-Tai
NT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D016959:Xanthomonas campestris / Q000378:metabolism*
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; C071567:general secretion pathway protein, Xanthomonas campestris