Serratia ATP-binding Cassette Protein Exporter, Lip, Recognizes a Protein Region Upstream of the C Terminus for Specific Secretion

Abstract

Serratia marcescens ATP-binding cassette (ABC) exporter, the Lip system, secretes lipase (LipASM), metalloproteases, and a cell surface layer protein homologue but not a heme acquisition protein, HasA (HasASM). Secretion of HasASM is limited to the HasSM system. However, HasA proteins from Pseudomonas fluorescens (HasAPF) and Pseudomonas aeruginosa were exported through the Lip and HasSM systems. To investigate the specificity in Lip exporter-mediated secretion, secretion analysis was performed using chimeras containing the HasA PF and HasASM sequences. The segment Val-Ala-Leu (designated R1 to R3 sites), which is present close to the C terminus of HasAPF but not HasASM, was revealed to be involved in the substrate specificity of the Lip exporter. Introduction of amino acid substitutions into the R1-R5 region demonstrated that R1, R3, R4, and R5 sites require some specific amino acid residues for Lip-mediated secretion. The amino acid sequence of the region was conserved considerably among the proteins secreted by the Lip exporter. On the contrary, the region was not related to HasA secretion through the HasSM system. Interestingly, a typical C-terminal motif, so far regarded as a secretion signal, was not necessary for secretion through either the Lip or the HasSM exporter. In LipA SM secretion via the Lip system, the typical C-terminal motif was not essential either, but the presence of a sequence similar to Val-Ala-Leu and its location from the C terminus greatly affect the secretion level. Secretion analyses using hybrid exporters and competitors exhibited that the R1-R5 region was recognized by an ABC protein of the Lip exporter, LipB, and that the mutations aborting Lip-mediated secretion in the region resulted in a loss of the affinity to LipB. Thus, a determinant within the secretory protein for Lip-mediated secretion was fully defined.