FIG 3.

Characterization of LapG specificity with the pCleevR assay. (A) Primary structure of pCleevR constructs in which various CdrA and LapA sequences containing their respective LapG cleavage sites are flanked N terminally by a His₆-tagged SUMO protein and C terminally by sfGFP A²²⁶R. (B) Length of the pCleevR sequence influences rate of cleavage by LapG. CdrA^short/LapA^short (as defined in panel A) sequences are cleaved more slowly than CdrA^long/LapA^long sequences, which are processed by LapG at approximately the same rate as CdrA^Cterm and LapA^Nterm. Cleavage products are indicated by asterisks (for CdrA) and number symbols (for LapA). Intact substrates are indicated with triangles. (C) Assessment of the relative rates of cleavage of CdrA^shortP′ and CdrA^shortP substrates compared with the CdrA^long substrate indicates that residues N terminal to the cleavage site are responsible for this increased rate of proteolysis. The fact that CdrA^randomP, which has the same length and number of residues flanking the cleavage site as CdrA^shortP′ but is of unrelated sequence, is cleaved more slowly than CdrA^shortP′ indicates that the enhanced rate is sequence dependent. The motif TAAG, in and of itself, is not sufficient for LapG targeting (right). Note that CdrA^long migrates with an aberrantly smaller size than CdrA^short in SDS-PAGE. (D) Single-amino-acid substitutions in CdrA^long identify residues D²⁰⁹⁷, P²⁰⁹⁸, and L²¹⁰¹ but not L²¹⁰⁰ as crucial for LapG-mediated cleavage. As with the TRRG variant of CdrA^Cterm (Fig. 2C), the analogous TRRG CdrA^long variant is not cleaved by LapG.