Mutations eliminating the protein export function of a membrane-spanning sequence.

Lee, Eungyung; Manoil, Colin

doi:10.1016/s0021-9258(19)61980-0

Cited by 49 publications

(4 citation statements)

References 52 publications

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“…1E, Table 1). While we cannot exclude that this enrichment is in part due to an increased focus on the transmembrane region in clinical research, we suggest—in line with previous work (63, 64)—that this observation also reflects a decreased mutational tolerance of the transmembrane region.…”

Section: Resultssupporting

confidence: 79%

Interpreting the molecular mechanisms of disease variants in human transmembrane proteins

Tiemann

Zschach

Lindorff‐Larsen

et al. 2022

Preprint

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Next-generation sequencing of human genomes reveals millions of missense variants, some of which may lead to loss of protein function and ultimately disease. We here investigate missense variants in membrane proteins -- key drivers in cell signaling and recognition. We find enrichment of pathogenic variants in the transmembrane region across 19,000 functionally classified variants in human membrane proteins. A key mechanism underlying pathogenicity in missense variants of soluble proteins has been shown to be loss of stability. We here perform structure-based estimations of changes in thermodynamic stability and evolutionary conservation analyses of 15 membrane proteins. We find evidence for loss of stability being the cause of pathogenicity in 59% of pathogenic variants, indicating that this is a driving factor also in membrane-protein-associated diseases. Our findings show how computational tools aid in gaining mechanistic insights into variant consequences for membrane proteins. To enable broader analyses of disease-related and population variants, we include variant mappings for the entire human proteome.

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Section: Resultssupporting

confidence: 79%

Interpreting the molecular mechanisms of disease variants in human transmembrane proteins

Tiemann

Zschach

Lindorff‐Larsen

et al. 2022

Preprint

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“…It is interesting to note in this context a recent paper by Lee and Manoil (1994). The authors showed that mutations eliminating the protein export function of a membranespanning sequence can be explained by a model where a minimum hydrophobicity is required for membrane insertion.…”

Section: Energetics Of Membrane Partitioning Of Proteinsmentioning

confidence: 99%

Thermodynamics of the Membrane Insertion Process of the M13 Procoat Protein, a Lipid Bilayer Traversing Protein Containing a Leader Sequence

et al. 1996

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For the first time, the standard free energy change, delta Gzero, of a membrane-inserting protein with a leader sequence has been determined experimentally, using M13 procoat protein as an example. The partition coefficient for the distribution of the procoat protein between the aqueous phase and the membrane phase of preformed lipid vesicles yielded a value of gamma = 6.5 x 10(5) M-1, corresponding to a delta Gzero of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC. For comparison, the partition coefficient of the M13 coat protein between the aqueous and the POPC lipid bilayer phase was determined to be distinctly lower: gamma = 1 x 10(5) M-1 (delta Gzero = -9.3 kcal/mol). Proteinase K digestion experiments have been performed, showing that 20% of the procoat protein bound to lipid vesicles spontaneously integrate in a transbilayer form, whereas 80% remain inserted in the interfacial membrane region. By taking together these results, an upper limit for the free energy change of the transmembrane insertion of procoat protein was estimated to be -14.8 kcal/mol. In order to distinguish further the contribution arising from insertion of the procoat protein into the membrane interfacial region from that due to transmembrane insertion, the partition coefficient of the mutant procoat protein OM30R [which contains a positively charged amino acid in its mature hydrophobic segment (exchange of a Val to an Arg residue at position 30)] was determined, yielding gamma = 0.3 x 10(5) M-1 (delta Gzero = -8.6 kcal/mol). Previously reported in vivo experiments have shown that the OM30R mutant protein is not translocated across Escherichia coli membranes but only binds to the inner surface. The results presented here indicate that although the insertion of the procoat protein into the interfacial region of the lipid bilayer contributes the major part to delta Gzero, it is the final energy gain of the interaction of the hydrophobic portions of the folded pre-protein with the lipid chains which drives the transmembrane insertion of the M13 procoat protein. Neither the leader sequence nor the mature coat protein alone yields this free energy gain. For the different proteins investigated here, spontaneous membrane insertion occurs only for fluid lipid bilayers, but not for membranes in the crystalline lipid phase. Furthermore, by using lipid bilayers with negative membrane surface charges, it was shown that both procoat and coat proteins are electrostatically attracted to the surface of the lipid membrane, though only to a small extent, with apparent partition coefficients of the same order of magnitude as for the phosphatidylcholine lipid membrane.

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“…MSII, with 14 consecutive hydrophobic amino acids, is completely integrated into the membrane while the parental protein, proOmpA, is fully translocated. Several analyses of the limits of variation of membrane-spanning segments (Zerial et al, 1987;MacIntyre et al, 1988;Kuroiwa et al, 1990Kuroiwa et al, , 1991Lee and Manoil, 1994;Chen and Kendall, 1995) have demonstrated that overall hydrophobicity, rather than the exact amino acid composition, determines the threshold for stop-transfer function (A.Sa ¨a ¨f, E.Wallin and G.von Heijne, submitted for publication). However, it was unknown whether this threshold defines the minimal hydrophobicity required to arrest translocation or whether it specifies exit from translocase and entry into the lipid bilayer.…”

Section: Discussionmentioning

confidence: 99%

Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function

Duong

1998

The EMBO Journal

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Preprotein translocase catalyzes membrane protein integration as well as complete translocation. Membrane proteins must interrupt their translocation and be laterally released from the translocase into the lipid bilayer. We have analyzed the translocation arrest and lateral release activities of Escherichia coli preprotein translocase with an in vitro reaction and the preprotein proOmpA carrying a synthetic stop-transfer sequence. Membrane protein integration is catalytic, occurs with kinetics similar to those of proOmpA itself and only requires the functions of SecYEG and SecA. Though a strongly hydrophobic segment will direct the protein to leave the translocase and enter the lipid bilayer, a protein with a segment of intermediate hydrophobicity partitions equally between the translocated and membrane-integrated states. Analysis of the effects of PMF, varied ATP concentrations or synthetic translocation arrest show that the stop-translocation efficiency of a mildly hydrophobic segment depends on the translocation kinetics. In contrast, the lateral partitioning from translocase to lipids depends solely on temperature and does not require SecA ATP hydrolysis or SecA membrane cycling. Thus translocation arrest is controlled by the SecYEG translocase activity while lateral release and membrane integration are directed by the hydrophobicity of the segment itself. Our results suggest that a greater hydrophobicity is required for efficient translocation arrest than for lateral release into the membrane.

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Mutations eliminating the protein export function of a membrane-spanning sequence.

Cited by 49 publications

References 52 publications

Interpreting the molecular mechanisms of disease variants in human transmembrane proteins

Interpreting the molecular mechanisms of disease variants in human transmembrane proteins

Thermodynamics of the Membrane Insertion Process of the M13 Procoat Protein, a Lipid Bilayer Traversing Protein Containing a Leader Sequence

Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function

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