Type II secretion apparatus of Xanthomonas campestris: Determination of ATPase activity of XpsE and mutant analysis of XpsE with mutations at nucleotide-binding motifs

• Main Authors: Ko-Min Kao, 高哿民
• Other Authors: Nien-Tai Hu
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/71992026939678427578

碩士 === 國立中興大學 === 生物化學研究所 === 93 === In the type II secretion pathway of Xanthomonas campestris pv. campestris, at least 12 protein products of the xps gene cluster are responsible for translocating extracellular proteins from periplasm to the milieu. Amoung them, the XpsE is the only protein without any membrane spanning sequence. It has four conserved nucleotide binding motifs, and is postulated to be an ATPase or a kinase to provide energy or signal for the secretion process. XpsL is a monotopic integral cytoplasmic membrane protein with its N-terminal (XpsLN) facing the cytoplasm. In this study, we observed XpsE displayed weak ATPase activity. The XpsE ATPase activity was stimulated slightly by MBP-XpsLN. Starch plate assay for extracellular -amylase indicates that single mutation at 5 conserved residues of XpsE abolished its in vivo function. We analyzed interaction between XpsE and XpsLN in vitro by performing maltose binding protein (MBP) pull-down assay. The wild type XpsE was pulled down by immobilized MBP-XpsLN, and the percentage bound to XpsLN of all mutant XpsE proteins are not much different from that of wild type XpsE. Determination of ATPase activity of XpsE indicated that single mutation K331M significantly diminished its ATPase activity, and E357Q mutation caused ATPase activity decrease by about half. The XpsL protein level, when expressed in the absence of other xps genes, was decreased by co-expressing with XpsE or XpsEK331M. In contrast, when co-expressing with XpsEE357Q or XpsEE395Q, XpsL protein level rose. To summary, while resulting in diminished ATPase activity, XpsEK331M remains capable of associating with XpsL, suggesting XpsE ATPase activity may function at a step after it associates with XpsL.