Research ArticleSTRUCTURAL BIOLOGY

Insertion and folding pathways of single membrane proteins guided by translocases and insertases

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Science Advances  30 Jan 2019:
Vol. 5, no. 1, eaau6824
DOI: 10.1126/sciadv.aau6824
  • Fig. 1 Unfolding fingerprint pattern of native LacY.

    (A) Schematics of the mechanical unfolding of native LacY from the phospholipid (PE/PG) membrane. The AFM stylus is pushed onto LacY (PDB 1PV7) to nonspecifically attach the elongated C-terminal end (polyGly LacY). Then, the cantilever is retracted to apply mechanical pulling force to the terminus. During retraction, LacY stepwise unfolds structural segments until being completely unfolded and extracted from the membrane (17). (B) Force-distance curve recorded upon unfolding a single LacY. The force-distance curve is shown as raw data (pale red) and smoothed (Savitzky-Golay filter, dark red). To obtain the contour lengths of mechanically unfolded polypeptide stretches (in amino acid), we fit every force peak using the worm-like chain (WLC) model (gray curves; Materials and Methods). (C) Density plot of 280 superimposed force-distance curves each showing the mechanical unfolding of one LacY. Mean contour lengths given at the top of each WLC curve define the ending of the previously unfolded structural segment and the beginning of the next segment to be unfolded. (D) Structural segments S1 to S10 mapped to the secondary structure of LacY as unfolded beginning from the C terminus. The C terminus is shown at the left, and transmembrane α-helices are numbered I to XII.

  • Fig. 2 YidC promotes stochastic insertion of LacY until having folded the native structure.

    (A) Schematic folding experiment of LacY in the presence of YidC. First, the AFM stylus attached to the C terminus is used to mechanically unfold and extract LacY from the membrane. Then, the unfolded LacY polypeptide is transported by the stylus in close proximity (~5 to 10 nm) to a phospholipid membrane embedding YidC. (B) After a folding time of 1 s, the stylus is retracted, recording a force-distance curve. To reveal whether the polypeptide folded structural segments, force peaks of the curve are fitted with the WLC model. WLC curves matching the fingerprint pattern of native LacY in terms of means ± SD are represented in black, and fits not matching are gray (fig. S6). (C) Probability of structural segments S1 to S10 inserting after 1-s folding time [number of structural segments (nss) = 231]. (D) Force-distance curve recorded in the presence of YidC after 2 s. (E) Probability of segments inserted after 2 s (nss = 266). (F) Force-distance curve recorded in the presence of YidC after 5 s. (G) Probability of segments inserted after 5 s (nss = 319). χ2 tests indicate (non)uniform distributions (***P < 0.001). NS, nonsignificant. Error bars indicate SE. More force-distance curves are shown in fig. S5.

  • Fig. 3 SecYEG promotes sequential insertion of LacY until having folded the native structure.

    (A) Schematic LacY folding experiment in the presence of SecYEG. An AFM stylus nonspecifically attached to the C terminus mechanically unfolds and extracts LacY from the membrane. The unfolded LacY polypeptide is then transported by the AFM stylus in close proximity (~5 to 10 nm) to a phospholipid membrane embedding SecYEG where it is kept for 1 s. (B) After this folding time, the AFM stylus is retracted, recording a force-distance curve. To reveal whether the polypeptide folded structural segments, force peaks of the curve are fitted with the WLC model. WLC curves matching the fingerprint pattern of native LacY in terms of means ± SD are represented in black, and fits not matching are gray (fig. S6). The example shows the insertion of structural segment S2. (C) Probability distribution of structural segments inserted after 1 s (nss = 271). (D) Force-distance curve recorded in the presence of SecYEG after 2 s. (E) Probability distribution of segments inserted after 2 s (nss = 114). (F) Force-distance curve recorded in the presence of SecYEG after 10 s. (G) Probability distribution of segments inserted after 10 s (nss = 198). χ2 tests indicate (non)uniform distributions (*P < 0.05, ***P < 0.001). Error bars indicate SE. More force-distance curves are shown in fig. S8.

  • Fig. 4 SecYEG-YidC fusion construct promotes sequential insertion of LacY until having folded the native structure.

    (A) Schematic LacY folding experiment in the presence of the SecYEG-YidC fusion construct. An AFM stylus nonspecifically attached to the C terminus mechanically unfolds and extracts LacY from the membrane. The unfolded LacY polypeptide is then transported by the AFM stylus in close proximity (~5 to 10 nm) to a phospholipid membrane embedding the SecYEG-YidC construct where it is kept for 1 s. (B) After this folding time, the AFM stylus is retracted, recording a force-distance curve. To reveal whether the polypeptide folded structural segments, force peaks of the curve are fitted with the WLC model. WLC curves matching the fingerprint pattern of native LacY in terms of means ± SD are represented in black, and fits not matching are gray (fig. S6). The example shows the insertion of structural segment S3. (C) Probability distribution of structural segments inserted after 1 s (nss = 206). (D) Force-distance curve recorded in the presence of SecYEG-YidC after 2 s. (E) Probability distribution of segments inserted after 2 s (nss = 384). (F) Force-distance curve recorded in the presence of SecYEG-YidC after 10 s. (G) Probability distribution of segments inserted after 10 s (nss = 301). χ2 tests indicate (non)uniform distributions (***P < 0.001). Error bars indicate SE. More force-distance curves are shown in fig. S9.

  • Fig. 5 In the presence of YidC and SecYEG, the translocase defines the folding pathway of the LacY polypeptide.

    (A) Folding kinetics of LacY in the presence of YidC, SecYEG, SecYEG-YidC fusion construct, or SecYEG and YidC from the SecYEG-YidC construct cleaved by the PreScission protease (fig. S10B). Colored linear fits approach the insertion and folding rate of structural segments. (B) Force-distance curves exemplify single LacY polypeptides inserting and folding 2, 3, or 4 neighbored structural segments in the presence of SecYEG. Force peaks matching the fingerprint pattern of native LacY are represented in black, and fits not matching are gray (fig. S6). (C) Probabilities of detecting the insertion of 2, 3, or 4 neighbored segments in the presence of YidC, SecYEG, SecYEG-YidC, or SecYEG and YidC. Four hundred seventy-eight experiments (force-distance curves) detecting insertion and folding events of LacY have been recorded in the presence of YidC, 395 in the presence of SecYEG, 397 in the presence of SecYEG-YidC fusion construct, and 313 in the presence of SecYEG and YidC. Statistical differences examined by analysis of covariance (A) and Z (C) tests were considered nonsignificant for P > 0.05 and significant for **P < 0.01 and ***P < 0.001. Error bars indicate SE.

  • Fig. 6 SecYEG and YidC insert and fold the membrane protein LacY along different pathways.

    (A) YidC starts insertion of the LacY polypeptide from any structural segment after which it stepwise inserts the remaining structural segments S1 to S10 of LacY. Structural segments insert in random order until folding of LacY has been completed. YidC offers 10! (3,628,800) pathways to fold the 10 structural segments S1 to S10 of LacY toward the native structure. (B) SecYEG alone or SecYEG and YidC together start insertion of the LacY polypeptide from any structural segment after which the remaining segments are inserted sequentially until folding of LacY has been completed. SecYEG or SecYEG and YidC offer 10 principal pathways to fold the 10 structural segments toward native LacY. Red dashed arrows indicate possibilities of initiating insertion. Red double arrows highlight the insertion and folding steps of the folding pathways. Black dashed arrows indicate the completion of the insertion and folding process.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau6824/DC1

    Fig. S1. LacY and YidC reconstituted into PE/PG phospholipid membranes.

    Fig. S2. SecYEG and SecYEG-YidC fusion construct reconstituted into PE/PG phospholipid membranes.

    Fig. S3. YidC, SecYEG, and SecYEG-YidC fusion constructs are functional in vivo.

    Fig. S4. Coadsorption of YidC and LacY proteoliposomes.

    Fig. S5. YidC facilitates random insertion and folding of LacY.

    Fig. S6. Classification criteria of LacY misfolding and folding events.

    Fig. S7. Completely unfolded and extracted LacY polypeptides cannot insert and fold into phospholipid membranes without assistance.

    Fig. S8. SecYEG facilitates sequential insertion and folding of LacY.

    Fig. S9. SecYEG and YidC together facilitate sequential insertion and folding of LacY.

    Fig. S10. SecY-YidC fusion construct cleaved by the PreScission protease.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. LacY and YidC reconstituted into PE/PG phospholipid membranes.
    • Fig. S2. SecYEG and SecYEG-YidC fusion construct reconstituted into PE/PG phospholipid membranes.
    • Fig. S3. YidC, SecYEG, and SecYEG-YidC fusion constructs are functional in vivo.
    • Fig. S4. Coadsorption of YidC and LacY proteoliposomes.
    • Fig. S5. YidC facilitates random insertion and folding of LacY.
    • Fig. S6. Classification criteria of LacY misfolding and folding events.
    • Fig. S7. Completely unfolded and extracted LacY polypeptides cannot insert and fold into phospholipid membranes without assistance.
    • Fig. S8. SecYEG facilitates sequential insertion and folding of LacY.
    • Fig. S9. SecYEG and YidC together facilitate sequential insertion and folding of LacY.
    • Fig. S10. SecY-YidC fusion construct cleaved by the PreScission protease.

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