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J. Biol. Chem., Vol. 280, Issue 6, 4585-4591, February 11, 2005

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Associations of the Major Pseudopilin XpsG with XpsN (GspC) and Secretin XpsD of Xanthomonas campestris pv. campestris Type II Secretion Apparatus Revealed by Cross-linking Analysis^*

Meng-Shiunn Lee, Ling-Yun Chen, Wei-Ming Leu, Rong-Jen Shiau¶, and Nien-Tai Hu¶||

From the Institute of Biotechnology and ^¶Institute of Biochemistry, National Chung Hsing University, Institute of Biochemistry, Chung Shan Medical University, 250 Kuo Kuang Road, Taichung 402, Taiwan, Republic of China

The major pseudopilin XpsG is an essential component of type II secretion apparatus of Xanthomonas campestris pv. campestris. Along with other ancillary pseudopilins, it forms a pilus-like structure spanning between cytoplasmic and outer membranes. Associations of pseudopilins with non-pseudopilin members of type II secretion apparatus were not well documented, probably due to their dynamic or unstable nature. In this study, by treating intact cells with a cleavable cross-linker dithiobis(succinimidylpropionate) (DSP), followed by metal chelating chromatography and immunoblotting on secretion-positive strains of X. campestris pv. campestris, we discovered associations of XpsGh with XpsN (GspC), as well as XpsD. These associations were detectable in a strain missing all components, but XpsO, of the type II secretion apparatus. However, chromosomal non-polar mutation in each gene exerted different effects upon the association between the other two. The XpsGh/XpsD association is undetectable in xpsN mutant; however, it was restored to a limited extent by overproducing XpsD protein. The XpsGh/XpsN association is unaltered by a lack of XpsD protein or an elevation of its abundance. Co-immune precipitation between XpsN and XpsD, while being independent of XpsG, was nonetheless enhanced by raising XpsG protein level. These observations agree with the proposition that the type II secretion apparatus in a cell may exist as an integrated multiprotein complex with all components working in concert. Moreover, in functional machinery, the association of the major pseudopilin XpsG with secretin XpsD appears strongly dependent on the existence of XpsN, the GspC protein.

Received for publication, August 16, 2004 , and in revised form, November 18, 2004.

^* This work was supported by Grant E-91-B-FA05-2-4 from the Ministry of Education and Grant NSC91-2311-B-005-039 from the National Science Council of the Republic of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Inst. of Biochemistry, National Chung Hsing University, 250 Kuo Kuang Rd., Taichung 402, Taiwan, Republic of China. Tel.: 886-4-22853486 (ext. 228); Fax: 886-4-22853487; E-mail: nthu{at}nchu.edu.tw.

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