Mechanism and hydrophobic forces driving membrane protein insertion of subunit II of cytochrome bo 3 oxidase

Abstract

Subunit II (CyoA) of cytochrome bo(3) oxidase, which spans the inner membrane twice in bacteria, has several unusual features in membrane biogenesis. It is synthesized with an amino-terminal cleavable signal peptide. In addition, distinct pathways are used to insert the two ends of the protein. The amino-terminal domain is inserted by the YidC pathway whereas the large carboxyl-terminal domain is translocated by the SecYEG pathway. Insertion of the protein is also proton motive force (pmf)-independent. Here we examined the topogenic sequence requirements and mechanism of insertion of CyoA in bacteria. We find that both the signal peptide and the first membrane-spanning region are required for insertion of the amino-terminal periplasmic loop. The pmf-independence of insertion of the first periplasmic loop is due to the loop's neutral net charge. We observe also that the introduction of negatively charged residues into the periplasmic loop makes insertion pmf dependent, whereas the addition of positively charged residues prevents insertion unless the pmf is abolished. Insertion of the carboxyl-terminal domain in the full-length CyoA occurs by a sequential mechanism even when the CyoA amino and carboxyl-terminal domains are swapped with other domains. However, when a long spacer peptide is added to increase the distance between the amino-terminal and carboxyl-terminal domains, insertion no longer occurs by a sequential mechanism.

Figure 1

Membrane topology of full length Pre-CyoA and the amino-terminal Pre-CyoA domain. The full-length Pre-CyoA is made with a cleavable signal peptide that is proteolytically removed by signal peptidase II processing. Pre-CyoA-N-P2 contains the Lep P2 domain added after transmembrane segment 1, allowing the construct to be immunoprecipitated using leader peptidase antiserum. SP represents the signal peptidase 2 cleavage site. The residues within transmembrane domain 1 and 2 were predicted from hydropathy plot and alkaline phosphatase fusions methods although the structure of the E. coli cytochrome bo oxidase suggests the transmembrane segment 1 is much longer beginning around residue 12.

Figure 2

Insertion of the amino-terminal domain of CyoA requires both the signal peptide and the membrane anchor domain 1. (a) Protease accessibility assay of the CyoA-N-P2 wild-type and single positive charge mutants. (b) Protease accessibility assay of CyoA-N-P2 double and triple positive charge mutants. DH5α cells expressing the indicated positive charge mutant of CyoA-N-P2 from plasmid pMS119 were grown to the mid-log phase and induced by the addition of 1 mM (final concentration) IPTG for 5 min. Samples were pulse-labeled with 50 mCi/ml [³⁵S]methionine for 2 min, and analyzed by the protease accessibility assay as described in Materials and Methods. The protein samples were immunoprecipitated using leader peptidase antiserum (which recognizes the P2 domain) and analyzed by SDS-PAGE and phosphorimaging. PK denotes proteinase K. GroEL, a cytoplasmic marker, and outer membrane protein A (OmpA), an outer membrane marker, are used as controls. G(-1)F preCyoA mutant was used as a precursor control. The lower band in the –PK lanes in Fig. 2a is a background band picked up with this batch leader peptidase antiserum. A different batch of antiserum was used in Fig. 2b.

Figure 3

The pmf-dependence of membrane insertion of the periplasmic domain is determined by the net charge of the amino-terminal domain. (a) The pmf stimulates membrane insertion of a negatively charged CyoA-N-P2 (K9D/R17D) mutant. (b) Pmf-independent translocation of periplasmic loop in the Pre-CyoA-N-P2 (WT). (c) The pmf impedes the membrane insertion of a positively charged CyoA-N-P2 (A4R/L5R) mutant. DH5α cells expressing the indicated mutant of CyoA-N-P2 from plasmid pMS119 were grown to mid-log phase and induced with 1 mM (final concentration) IPTG for 5 min. Cultures were then labeled with 50 mCi/ml [³⁵S]methionine for 2 min and analyzed by the protease accessibility assay. Where indicated, the pmf was abolished by treatment with CCCP (50 µM final concentration) for 45 s after IPTG induction. The protein samples were immunoprecipitated and analyzed as in Figure 2. PK denotes proteinase K. The lower band in the –PK lanes (Fig. 2a and b) is a background band picked up with the batch of leader peptidase antiserum. The position of the precursor form of CyoA was determined by accumulation of the precursor by growth of YidC-depletion strain, JS7131, bearing either the CyoA-N-P2 (K9D/R17D) or CyoA-N-P2 (WT) in glucose.

Figure 4

Translocation of the large carboxyl-terminal periplasmic domain of CyoA requires insertion of the amino-terminal domain. (a) Introduction of two positively charged residues into membrane anchor domain I inhibits membrane insertion. (b) Introduction of two positively charged residues into the signal peptide inhibits membrane insertion. (c) A deletion within the signal peptide blocks membrane insertion of CyoA. (d) Pmf-dependent insertion of the carboxyl-terminal domain of CyoA containing acidic residues in the amino-terminal domain. (e). Slight pmf-dependent insertion of the CyoA carboxyl-terminal domain and pmf-independent insertion of the amino-terminal domain of the wild-type CyoA. JS7131 cells bearing the plasmid pMS119 containing the indicated positively charged CyoA-His₁₀ construct were grown under YidC expression (Ara) or depletion (Glc) conditions, the protein was induced with 1 mM IPTG for 5 min, pulse-labeled with 50 µCi/ml [³⁵S] methionine for 2 min and analyzed for membrane insertion by the protease accessibility assay, as described in Materials and Methods. One JS7131 culture was treated without IPTG induction. DH5α cells expressing the indicated signal peptide deletion mutant of CyoA, Pre-CyoA (-5), or Pre-CyoA (WT) from plasmid pMS119 were grown to the mid-log phase and induced by the addition of 1 mM (final concentration) IPTG for 5 min. Samples were pulse-labeled with 50 mCi/ml [³⁵S]methionine for 2 min, and analyzed by the protease accessibility assay as described in Materials and Methods. Where indicated, the pmf was abolished in DH5á cells by treatment with CCCP (50 µM) for 45 s after IPTG induction. The protein samples were then either acid-precipitated or pulled down using BD TALON™ metal affinity resin when a His-tag was present, as described in Materials and Methods and analyzed by SDS-PAGE and phosphorimaging. PK denotes proteinase K.

Figure 4

Translocation of the large carboxyl-terminal periplasmic domain of CyoA requires insertion of the amino-terminal domain. (a) Introduction of two positively charged residues into membrane anchor domain I inhibits membrane insertion. (b) Introduction of two positively charged residues into the signal peptide inhibits membrane insertion. (c) A deletion within the signal peptide blocks membrane insertion of CyoA. (d) Pmf-dependent insertion of the carboxyl-terminal domain of CyoA containing acidic residues in the amino-terminal domain. (e). Slight pmf-dependent insertion of the CyoA carboxyl-terminal domain and pmf-independent insertion of the amino-terminal domain of the wild-type CyoA. JS7131 cells bearing the plasmid pMS119 containing the indicated positively charged CyoA-His₁₀ construct were grown under YidC expression (Ara) or depletion (Glc) conditions, the protein was induced with 1 mM IPTG for 5 min, pulse-labeled with 50 µCi/ml [³⁵S] methionine for 2 min and analyzed for membrane insertion by the protease accessibility assay, as described in Materials and Methods. One JS7131 culture was treated without IPTG induction. DH5α cells expressing the indicated signal peptide deletion mutant of CyoA, Pre-CyoA (-5), or Pre-CyoA (WT) from plasmid pMS119 were grown to the mid-log phase and induced by the addition of 1 mM (final concentration) IPTG for 5 min. Samples were pulse-labeled with 50 mCi/ml [³⁵S]methionine for 2 min, and analyzed by the protease accessibility assay as described in Materials and Methods. Where indicated, the pmf was abolished in DH5á cells by treatment with CCCP (50 µM) for 45 s after IPTG induction. The protein samples were then either acid-precipitated or pulled down using BD TALON™ metal affinity resin when a His-tag was present, as described in Materials and Methods and analyzed by SDS-PAGE and phosphorimaging. PK denotes proteinase K.

Figure 5

Addition of a long linker after membrane anchor 1 of Pre-CyoA allows insertion to proceed by a non-sequential mechanism, but a sequential mechanism still occurs with different amino-terminal and carboxyl-terminal domains. YidC depletion studies with constructs CyoA-P₁₀ (a), CyoA (WT) (b), PC-C-CyoA (c) and N-CyoA-Lep (d). JS7131 cells bearing the plasmid pMS119 with the indicated construct were grown under YidC expression (Ara) or depletion (Glc) conditions, the protein was induced with 1 mM IPTG for 5 min, pulse-labeled with 50 µCi/ml [³⁵S]-methionine for 2 min and analyzed for membrane insertion by the protease accessibility assay, as described in Materials and Methods. The protein samples were then either acid-precipitated (CyoA, top panel) or immunoprecipitated using GroEL and OmpA antisera (bottom panel) and analyzed by SDS-PAGE and phosphorimaging.

Figure 5

Addition of a long linker after membrane anchor 1 of Pre-CyoA allows insertion to proceed by a non-sequential mechanism, but a sequential mechanism still occurs with different amino-terminal and carboxyl-terminal domains. YidC depletion studies with constructs CyoA-P₁₀ (a), CyoA (WT) (b), PC-C-CyoA (c) and N-CyoA-Lep (d). JS7131 cells bearing the plasmid pMS119 with the indicated construct were grown under YidC expression (Ara) or depletion (Glc) conditions, the protein was induced with 1 mM IPTG for 5 min, pulse-labeled with 50 µCi/ml [³⁵S]-methionine for 2 min and analyzed for membrane insertion by the protease accessibility assay, as described in Materials and Methods. The protein samples were then either acid-precipitated (CyoA, top panel) or immunoprecipitated using GroEL and OmpA antisera (bottom panel) and analyzed by SDS-PAGE and phosphorimaging.