Article Figures & Data
Figures
- Figure 1. MPIase is involved in membrane insertion of Pf3-Lep (amber).
(A) Membrane topologies of Pf3-Lep (amber) (left) and its V15D mutant (right). The N-terminal region is exposed to the periplasm and the C-terminal region to the cytosol. Negatively charged residues in the periplasmic region are denoted by red dots. (B) Preparation of MPIase- and YidC-depleted INV. Depletion of MPIase (upper panel) and YidC (lower panel) was confirmed by immunoblotting. Note that MPIase is up-regulated by YidC depletion. The relative levels of MPIase and YidC are shown below the blots. Whole cell extract (1 μg protein) and INV (10 μg protein) were used to detect MPIase and YidC, respectively. (C) Schematic representation of assaying of membrane insertion in vitro. A membrane-protected fragment (MPF) arises upon PK digestion. The C-terminal region of Pf3-Lep (amber) is digested, giving the MPF. (D) Membrane insertion of both Pf3-Lep (amber) and V15D is stimulated by signal recognition particle. The substrates were in vitro synthesized in the presence of INV prepared from EK413 (WT) as specified. Upon PK digestion after synthesis, three bands, “i,” “c,” and “r,” appeared. Band “i” represents incomplete insertion, while band “c” represents the MPF. Band “r” is the PK-resistant band because this non-specifically appeared even in the absence of membranes. Hereafter, this band is indicated by asterisks. The insertion activity (the percentage of the level of “c” as to that of the substrates) is shown below the autoradiograms. The numbers of methionine (three in the substrates and two in MPF) were considered in the calculation. The positions of molecular weight markers are also shown by dots. (E) Membrane insertion of Pf3-Lep (amber) and V15D into MPIase- and YidC-depleted INV. (B) The substrates were in vitro synthesized in the presence of ΔMPIase INV and ΔYidC INV, shown in (B), as specified. (D) The insertion activity was determined as described in (D) and is shown.
Source data are available for this figure.
Source Data for Figure 1[LSA-2021-01162_SdataF1.pdf]
- Figure S1. Amino acid sequence and membrane topologies of the substrates used in this study.
(A) Amino acid sequences of Pf3-Lep (amber) and V15D. The N-terminal region (light brown) was derived from Pf3 coat, while the C-terminal region (blue) was derived from TM1 and the subsequent cytosolic region of the leader peptidase. Acidic amino acids are denoted in red and basic amino acids in green. (B) Membrane topologies of Pf3-Lep (amber) and V15D.
- Figure 2. Effects of SecA and proton motive force (PMF) on Pf3-Lep (amber) and V15D insertion.
(A) Effect of sodium azide on SecA-dependent pOmpA translocation. pOmpA was in vitro synthesized in the presence of INV prepared from EK413, followed by the PK protection assay. Sodium azide (1 mM) was added as specified. The percentage of the translocated materials (pOmpA plus OmpA) is shown at the bottom. The numbers of methionine (six in pOmpA and five in OmpA) were considered in the calculation. (B) Effect of DCCD on PMF-dependent stimulation of Pf3 coat insertion. Pf3 coat was in vitro synthesized in the presence of INV prepared from EK413, followed by the PK protection assay. DCCD (0.15 mM) or DMSO was added as specified. The insertion activity was determined and is shown at the bottom. (C) Effect of sodium azide on Pf3-Lep (amber)/V15D insertion. The insertion activity for Pf3-Lep (amber) (left half) and V15D (right half) was determined as described in the legend to Fig 1C, and is shown at the bottom. (A) Where specified, sodium azide was added as in (A). The position of membrane-protected fragment is indicated. The PK-resistant bands that appeared in the absence of INV as indicated by asterisks. (D, E) Effect of DCCD on Pf3-Lep (amber)/V15D insertion. The insertion activity for Pf3-Lep (amber) (left half) and V15D (right half) was determined as described in the legend to Fig 1D, and is shown at the bottom. (B) DCCD was added as in (B). (D, E) INV prepared from EK413 were used in (D), whereas ΔYidC or YidC+ INV prepared from JS7131 were used in (E) as specified. The position of membrane-protected fragment is indicated. The PK-resistant bands unrelated with membrane insertion are indicated by asterisks.
- Figure 3. Reconstitution of Pf3-Lep (amber) and V15D insertion.
(A) Spontaneous insertion of both Pf3-Lep (amber) and V15D is blocked by a physiological level of DAG. Pf3-Lep (amber) (left half) and V15D (right half) were in vitro synthesized in the presence of liposomes formed with phospholipids (PL) or phospholipids and DAG (PL+DAG), followed by the PK protection assay. The insertion activity was determined and is shown at the bottom. The position of membrane-protected fragment is indicated. The PK-resistant bands unrelated with membrane insertion are indicated by asterisks. (B) Effects of MPIase and YidC on Pf3-Lep (amber) and V15D insertion. (Proteo)liposomes containing MPIase and YidC were reconstituted, followed by assaying of Pf3-Lep (amber) (upper panel) and V15D (lower panel) insertion. The insertion activity was determined as described in the legend to Fig 1D and is shown at the bottom. (C) Effects of PMF on Pf3-Lep (amber) and V15D insertion. F0F1-ATPase was co-reconstituted with MPIase and YidC to impose PMF. The proteoliposomes thus reconstituted were subjected to Pf3-Lep (amber) and V15D insertion. The insertion activity was determined and is shown at the bottom. In all the autoradiograms, the position of membrane-protected fragment is indicated. Also, the PK-resistant bands unrelated with membrane insertion are indicated by asterisks.
- Figure 4. Expression level of the substrates affects the dependency on insertion factors.
(A, B) Pf3-Lep (amber) and V15D insertion into proteoliposomes becomes dependent on YidC and proton motive force as their expression levels increase. (A) The insertion reactions were carried out in the presence of 10 MBq radioactive methionine/ml to increase the expression levels of Pf3-Lep (amber) (upper panel) and V15D (lower panel) (A). (B) The expression levels were further increased by adding cold methionine (0.3 mM) (B). The insertion activity for each lot of proteoliposomes was determined as described in the legend to Fig 1D, shown at the bottom. The position of membrane-protected fragment is indicated. The PK-resistant bands unrelated with membrane insertion are indicated by asterisks. The experiments were carried out at least three times. Average activities with error bars are shown at the right of each autoradiogram.
- Figure 5. MPIase and YidC interact directly.
(A) Expression level of YidC in INV (10 μg protein) prepared from JS7131 and JS7131/pTac-YidC-CHis. It was analyzed by immunoblotting using anti-YidC antibody. (B) Purification of YidC on TALON column chromatography. Solubilized membranes of each INV were applied onto the TALON column, followed by elution with 150 mM imidazole. The eluted fraction (5 μl) was subjected to immunoblotting using anti-YidC antibody. (C) Detection of MPIase in the eluted fractions. (B) The eluted fractions in (B) (5 μl) were analyzed on TLC, and then visualized by immunostaining using anti-MPIase antibody. Purified MPIase (10 ng) was used as a standard. The positions of TLC origin/front and MPIase are shown.
Source data are available for this figure.
Source Data for Figure 5[LSA-2021-01162_SdataF5.pdf]
- Figure 6. Current model for Pf3-Lep (amber) and V15D insertion into membranes.
When the expression levels of substrates are low, MPIase is sufficient for insertion (left). As the expression levels of the substrates increase, the MPIase-dependent insertion being to be stimulated by YidC and proton motive force. The yellow arrow represents the generation of proton motive force. The negatively charged residues in the periplasmic region of Pf3-Lep and V15D are denoted by red dots. See the text for details.
Tables
- Table 1.
Insertion factor dependency for membrane proteins determined in the in vitro reconstitution system.
Protein SecYEG MPIase YidC Proton motive force References MtlA Essential Essential Conditional No effect 6 and 7 Pf3 coat Not essential Essential Stimulate Stimulate 7 and 22 3L-Pf3 coat Not essential Essential Conditional No effect 7, 15, 16, and 22 M13 procoat Not essential Essential Stimulate Stimulate 6, 7, and 14 F0c Not essential Essential Stimulate No effect 8, 9, and 23 Pf3-Lep Not essential (This study) Not required No effect 24 Pf3-Lep V15D Not essential (This study) Stimulate Stimulate 24 The insertion factor dependencies for specified membrane proteins are indicated. “Conditional” denotes that YidC stimulates insertion when the substrate level is high. In the case of Pf3-Lep and Pf3-Lep V15D, used in this study, the in vivo results are indicated.
