Analyse der subzellulären Lokalisation des C-Signalvorläuferproteins p25 und die Identifikation der PopC-Spaltstelle in p25 in Myxococcus xanthus

Myxococcus xanthus is a Gram-negative, rod-shaped δ-proteobacterium, which shows a complex developmental program. This program is initiated under starvation conditions and results in the formation of multicellular, spore-filled fruiting bodies. After six hours of starvation the C-signal coordinates and regulates the aggregation of cells into fruiting bodies, sporulation of cells into myxospores and a specific gene expression. The C-signal is a 17 kDa protein p17, which is generated by proteolytic cleavage of a 25 kDa precursor p25 by the subtilisin-like serine-protease PopC. The aim of this study was to investigate the subcellular localization of p25 in the outer membrane and to identify the PopC cleavage site in p25. To analyze the localization of p25, intact cells were treated with an unspecific protease. Immunoblot analysis with antibodies against p25 and against different control proteins were used to detect protein degradation. p25 was degraded like the outer membrane protein PilQ, which is exposed to the cell surface. Additionally, the outer membrane protein Tgl, which is exposed into the periplasmic space, and the inner membrane protein PilC were employed as controls. Tgl and PilC were degraded less drastically than PilQ and p25. These data suggest that p25 is exposed on the cell surface. To identify the PopC cleavage site, biochemical and genetic approaches were used. Synthetic peptides were explored to map the PopC cleavage site and the N-terminus of p17 was analyzed by mass-spectrometry. Truncated MalE-p25 derivatives as well as mutants containing alanine substitutions at certain amino acid positions were investigated after cleavage by purified PopC protein in vitro. With these experiments the PopC cleavage site was narrowed down to the amino acid motif 56LDV58 in p25. Furthermore, analysis of different alanine substitution mutants in vitro and in vivo revealed that aspartate in the position 57 of p25 is essential for PopC cleavage in M. xanthus. These data strongly suggest that PopC cleaves p25 after aspartate 57 in the cleavage motif 56LDV58.