FIGURE 3: Analyses of reporter proteins in wild-type and formylation-lacking fmt E. coli.

(A) Design of the P1^T2 (D2^1-11-eK-ha-Ura3) reporter protein. The term P1^T2 (protein-1 containing Thr at position 2) denotes a chimeric reporter containing the indicated N‑terminal segments upstream of the 267-residue S. cerevisiae Ura3 moiety. Arrowheads indicate deformylation of N-terminal fMet by the PDF deformylase and the subsequent removal of Met by MetAP. See the main text for details.

(B) Same as in (A) but the otherwise identical P1^T2D reporter contains Asp (D) at position 2. The rate of PDF-mediated deformylation of N-terminal fMet with Asp at position 2 is at least 10-fold lower than the rate of deformylation with Thr at position 2. Another difference between P1^T2 and P1^T2D is the retention of N-terminal Met in P1^T2D. See the main text for details and citations.

(C) The use of ubiquitin (Ub) fusions to generate P1^T2 and P1^T2D through the removal of the N-terminal Ub moiety by the S. cerevisiae Ubp1 deubiquitylase expressed in E. coli.

(D) Immunoblotting analyses, after SDS-PAGE, of the P1^T2 reporter protein expressed in fmt (lane 1), wild-type (lane 2) and fmt def (lane 3) E. coli. Lanes 4-6, the corresponding total protein patterns (Coomassie staining). Lane 7, molecular mass markers.

(E) Immunoblotting analyses, after SDS-PAGE, of the P1^T2 and P1^T2D reporters expressed from the P_ara promoter in wild-type E. coli (lanes 1-4), and of the Ub fusions Ub-P1^T2 and Ub-P1^T2D in wild-type E. coli expressing the S. cerevisiae Ubp1 deubiquitylase (lanes 5-8).

(F) Same as in (E), but independent experiments, in addition to expressing Ub-P1^T2 and Ub-P1^T2D in the absence of coexpressed yeast Ubp1 (lanes 9-12). See the main text for details and citations.