Figure 2. Expression and topology of mutant LmrP proteins.

A, Western blot of total membrane proteins in inside-out membrane vesicles (ISOVs) (15 µg of protein/lane) probed with anti-His₅-tag antibody, showing equal expression of Wt and mutant LmrP proteins in the plasma membrane of L. lactis NZ9000 ΔlmrA ΔlmrCD (with an apparent molecular mass on 10% SDS-PAGE of about 32 kDa), and lack of this expression in controls cells. B, The availability of the C-terminal His-tag in Wt LmrP, and the K357A and R260A mutants to cleavage by proteinase K (+ PK) at the external side of ISOVs and right-side-out membrane vesicles (RSOVs) was tested during incubations of 10, 20 and 30 min. Subsequently, remaining C-terminal His-tag was detected by Western blotting. Control incubation (lane 1, -PK) is without proteinase K. Equal loading of the lanes was confirmed by densitometry of the same samples on Coomassie-stained SDS-PAGE (not shown).