Figure 2.
Figure 2. Tat-dependent translocation of reporter proteins fused to the transmembrane domains of the S. coelicolor Rieske protein. (A) Schematic representation of the constructs used in these experiments. Amino acid numbers are indicated. (B) Tat-dependent translocation of MBP fused to the hydrophobic portion of Sco2149. E. coli strain HS3018-A (deleted for malE) and an otherwise isogenic tatABC mutant strain containing either pBAD24 (vector), pBAD24 producing the Sco2149-MBP fusion protein (TM123-MBP), or variants of this construct where the twin arginine motif before TM3 or the lysine-arginine motif before TM1 were substituted to twin lysines (TM12KK3-MBP or KKTM123-MBP, respectively) were cultured on maltose indicator broth containing bromocresol purple, as described in Materials and methods. (C) Tat-dependent translocation of AmiA fused to the hydrophobic portion of Sco2149. E. coli strain MCDSSAC (which carries chromosomal deletions in the signal peptide coding regions of amiA and amiC) and its cognate tatABC mutant containing either pSU18 (vector), pSU18 producing native AmiA (AmiA), pSU18 producing the Sco2149-AmiA fusion protein (TM123-AmiA), or variants of this construct where the twin arginine motif before TM3 was substituted to twin lysines or where the native AmiA signal peptidase cleavage site was present (TM12KK3-AmiA or TM123-(AXA)AmiA, respectively) were cultured on LB medium containing 1% SDS as described in Materials and methods. (D) Proteinase K accessibility of the TM123-MBP fusion protein in right-side-out membrane vesicles. Sphaeroplasts were prepared from HS3018-A or HS3018-AΔtatABC strains producing TM123-MBP, TM12KK3-MBP, or KKTM123-MBP. Samples were treated with proteinase K, precipitated with TCA, and analyzed via immunoblotting using anti-MBP antiserum. The numbers beneath the lanes are the percentage of full-length fusion protein remaining after proteinase K treatment (±SD, where n = 6 for the TM123-MBP samples and n = 3 for the TM12KK3-MBP and KKTM123-MBP samples). (E) Proposed topologies of TM123-MBP in tat+ and tat- strains of E. coli. The arrow indicates a probable protease accessible site in the periplasmic loop region between TM1 and TM2. (F) TM3 of Sco2149 alone can act as a Tat-targeting sequence. E. coli strain HS3018-A and an isogenic tatABC mutant containing either pBAD24 (vector) or pBAD24 producing variants of a Sco2149-MBP fusion protein containing an initiating methionine, and then the amino acids of Sco2149 (130–186 [130-MBP], 148–186 [148-MBP], 154–186 [154-MBP], and 158–186 [158-MBP]) fused to the mature region of MBP were cultured on maltose indicator broth containing bromocresol purple as described in Materials and methods. As an additional control, the twin arginine motif of construct 158-MBP was mutated to twin lysine (158KK-MBP) and also tested for the ability to support metabolism of maltose.

Tat-dependent translocation of reporter proteins fused to the transmembrane domains of the S. coelicolor Rieske protein. (A) Schematic representation of the constructs used in these experiments. Amino acid numbers are indicated. (B) Tat-dependent translocation of MBP fused to the hydrophobic portion of Sco2149. E. coli strain HS3018-A (deleted for malE) and an otherwise isogenic tatABC mutant strain containing either pBAD24 (vector), pBAD24 producing the Sco2149-MBP fusion protein (TM123-MBP), or variants of this construct where the twin arginine motif before TM3 or the lysine-arginine motif before TM1 were substituted to twin lysines (TM12KK3-MBP or KKTM123-MBP, respectively) were cultured on maltose indicator broth containing bromocresol purple, as described in Materials and methods. (C) Tat-dependent translocation of AmiA fused to the hydrophobic portion of Sco2149. E. coli strain MCDSSAC (which carries chromosomal deletions in the signal peptide coding regions of amiA and amiC) and its cognate tatABC mutant containing either pSU18 (vector), pSU18 producing native AmiA (AmiA), pSU18 producing the Sco2149-AmiA fusion protein (TM123-AmiA), or variants of this construct where the twin arginine motif before TM3 was substituted to twin lysines or where the native AmiA signal peptidase cleavage site was present (TM12KK3-AmiA or TM123-(AXA)AmiA, respectively) were cultured on LB medium containing 1% SDS as described in Materials and methods. (D) Proteinase K accessibility of the TM123-MBP fusion protein in right-side-out membrane vesicles. Sphaeroplasts were prepared from HS3018-A or HS3018-AΔtatABC strains producing TM123-MBP, TM12KK3-MBP, or KKTM123-MBP. Samples were treated with proteinase K, precipitated with TCA, and analyzed via immunoblotting using anti-MBP antiserum. The numbers beneath the lanes are the percentage of full-length fusion protein remaining after proteinase K treatment (±SD, where n = 6 for the TM123-MBP samples and n = 3 for the TM12KK3-MBP and KKTM123-MBP samples). (E) Proposed topologies of TM123-MBP in tat+ and tat- strains of E. coli. The arrow indicates a probable protease accessible site in the periplasmic loop region between TM1 and TM2. (F) TM3 of Sco2149 alone can act as a Tat-targeting sequence. E. coli strain HS3018-A and an isogenic tatABC mutant containing either pBAD24 (vector) or pBAD24 producing variants of a Sco2149-MBP fusion protein containing an initiating methionine, and then the amino acids of Sco2149 (130–186 [130-MBP], 148–186 [148-MBP], 154–186 [154-MBP], and 158–186 [158-MBP]) fused to the mature region of MBP were cultured on maltose indicator broth containing bromocresol purple as described in Materials and methods. As an additional control, the twin arginine motif of construct 158-MBP was mutated to twin lysine (158KK-MBP) and also tested for the ability to support metabolism of maltose.

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