The Mgm1p model protein. (A) Alternative topogenesis of Mgm1p in the mitochondrial inner membrane (Herlan et al, 2004). Mgm1p is imported through the TIM23 translocon. The presequence (black) is cleaved by the matrix processing peptidase (MPP). The first hydrophobic segment (red) integrates into the membrane in about half of the molecules (left, l-Mgm1p). In the remaining molecules, the first hydrophobic segment translocates into the matrix, leaving the second hydrophobic segment (blue) spanning the membrane (right). The second segment is cleaved by the inner membrane protease Pcp1p, giving rise to s-Mgm1p. (B) The first hydrophobic segment of Mgm1p (residues 87–113, italics) was replaced by 37-residue long segments including an H-segment, its immediate flanking residues (GGPG…GPGG), and five additional flanking residues from H-segment constructs previously analysed in the ER (underlined).

Membrane insertion efficiency of Leu/Ala-based H-segments. (A) Whole-cell lysates from yeast transformants expressing HA-tagged Mgm1p constructs with H-segments containing varying number of Leu residues were analysed by SDS–PAGE and western blotting. l-Mgm1p is the membrane-anchored long isoform and s-Mgm1p is the soluble short isoform. Three examples of H-segments are shown below the gel; all H-segment sequences are listed in Supplementary Table S1. (B) Membrane insertion efficiency expressed as ΔG_app plotted against the number of Leu residues in the H-segment. Averages from at least three independent experiments and standard errors are shown. (C) Western blot of in vitro-translated HA-tagged Mgm1p precursor (lane 1) and Mgm1p(6L/13A) precursor (lane 4), and of whole-cell lysate samples from yeast transformants expressing HA-tagged Mgm1p (lane 2) and Mgm1p(6L/13A) (lane 3). p-Mgm1p, l-Mgm1p, and s-Mgm1p denote, respectively, the precursor form, the long mature isoform, and the short mature isoform of HA-tagged Mgm1p.

‘Biological' ΔG^X_app scales. (A) The mitochondrial ΔG_app^X scale derived from Leu/Ala-based H-segments with the indicated amino acid placed in the middle of the H-segment (see Supplementary Table S1). Averages from at least three independent experiments and standard errors are shown. (B) Comparison between the ΔG_app^X scales obtained for the mitochondrial inner membrane and for the mammalian ER membrane (Hessa et al, 2007).

Position-dependent effects of different amino acids on membrane insertion. (A) Symmetric pair-scan for Pro residues in the mitochondrial inner membrane (circles) and the mammalian ER membrane (squares). (B) Single-Pro scan of H-segment insertion into the mitochondrial inner membrane (circles) and the mammalian ER membrane (squares). The ΔG_app value for the 6L/13A H-segment in the mitochondrial inner membrane is shown as a strikethrough circle. (C) Symmetric pair-scans for Trp (blue), Phe (green), and Tyr (red). Dashed lines show the corresponding data for the mammalian ER. The ΔG_app value (mitochondrial inner membrane) for the 3L/16A (green), 4L/15A (blue), and 5L/14A (red) H-segments are shown as strikethrough circles. (D) Lys (blue) and Asp (red) scans. Dashed lines show the corresponding data for the mammalian ER. The ΔG_app value (mitochondrial inner membrane) for the 6L/13A H-segment is shown as a strikethrough circle. In all panels, data points are averages from at least three independent experiments and standard errors are shown.

Effects of charged flanking residues on membrane insertion. Flanking sequences have the general design XXPX…XPXX (denoted 3X-3X) where X=G, R, K, or D. The results are expressed as ΔΔG_app values relative to the corresponding construct with GGPG…GPGG flanks. For the 3K-3G and 3G-3K flanks, the ΔΔG_app values have been averaged over multiple H-segments of different composition (see Supplementary Table S1). Averages from at least three independent experiments and standard errors are shown.

Analysis of H-segments in the context of the CoxVa protein. (A) CoxVa has an N-terminal mitochondrial-targeting peptide that is cleaved upon import by the matrix-localized mitochondrial processing peptidase (MPP). A segment consisting of residues 96–122 (containing the single-transmembrane helix in CoxVa) was replaced by GGPG…GPGG-flanked H-segments. If the H-segment is not inserted into the inner membrane but translocated to the matrix, it is protected from proteinase K (PK) treatment, whereas if it is inserted, the C-terminal end will be exposed to the IMS and therefore accessible to PK. (B) CoxVa constructs with the indicated H-segments were synthesized in a cell-free translation system in the presence of [³⁵S] methionine and imported into yeast mitochondria. Mitoplasts were prepared by swelling (SW), treated with PK and analysed by SDS–PAGE. A control import reaction was also performed in the presence of valinomycin to dissipate the membrane potential (ΔΨ). Precursor protein is indicated by a blue arrow, full-length mature protein resulting from MPP cleavage by a red arrow, and PK-trimmed protein by a yellow arrow. Quantitation of the amounts of non-inserted (i.e. translocated), full-length mature protein is shown below the gel images (with standard deviations). (C) Amounts of non-inserted, full-length mature protein for 3L/16A H-segments with GGPG…DPDD, GGPG…KPKK, DDPD…GPGG, and KKPK…GPGG flanks. Averages from at least three independent experiments and standard errors are shown.

The mitochondrial import motor affects membrane insertion. (A) Wild-type Mgm1p and two Mgm1p constructs carrying H-segments GGPG-5L/14A-GPGG and DDPD-8L/11A-GPGG were expressed in the temperature-sensitive pam16-3 mutant strain and in the isogenic PAM16 wild-type strain. Yeast transformants were grown at 30°C, and whole-cell lysates were subjected to TCA precipitation followed by SDS–PAGE and western blotting using an anti-HA antibody. The long and short Mgm1p isoforms are indicated. The 5L/14A and D-8L/11A-G lanes are from simultaneous experiments and were assembled from non-neighbouring lanes on the gel. (B) ΔG_app values for Mgm1p constructs carrying the indicated H-segments. White circles are for the pam16-3 strain and black circles for the corresponding isogenic wild-type strain. Averages from at least three independent experiments and standard errors are shown.

Mean frequencies of positively (K+R, top panel) and negatively (D+E, bottom panel) charged residues in the 10 residues flanking the transmembrane segments on the matrix side (grey bars), in the transmembrane segment (white bars), and in the 10 residues flanking the transmembrane segment on the intermembrane space (IMS) side (black bars) in a collection of transmembrane segments in mitochondrial proteins imported via a ‘conservative' sorting mechanism in which the protein is first fully imported into the matrix and then inserted into the inner membrane from the matrix side (eight proteins, see Materials and methods), or via a ‘stop-transfer' mechanism in which the transmembrane segment is arrested in the TIM23 translocon and inserted laterally into the inner membrane (21 proteins). Standard errors are shown.