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The surfactant peptide KL4 sequence is inserted with a transmembrane orientation into the endoplasmic reticulum membrane.

ABSTRACT: Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic surfactant peptide KL(4), a peptide based on a C-terminal amphipathic helical region of human SP-B, efficiently mimics some functional properties of SP-B and is included in therapeutic surfactant preparations used in trials to treat respiratory distress syndrome. The membrane orientation of this peptide is controversial. We used an in vitro transcription-translation system to study the insertion of hydrophobic sequences into microsomal membranes, and showed that the KL(4) sequence integrates efficiently with a transmembrane orientation despite the presence of intermittent lysines throughout the sequence. In contrast, the precise sequence of the C-terminal SP-B amphipathic region failed to integrate, indicating a nontransmembrane orientation. Differences in the membrane insertion between KL(4) and the SP-B-inspiring sequence match predictions from calculated free energies of insertion of the two sequences into membranes.

SUBMITTER: Martinez-Gil L

PROVIDER: S-EPMC2527282 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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The surfactant peptide KL4 sequence is inserted with a transmembrane orientation into the endoplasmic reticulum membrane.

Martínez-Gil Luis L Pérez-Gil Jesús J Mingarro Ismael I

Biophysical journal 20080711 6

Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic surfactant peptide KL(4), a peptide based on a C-terminal amphipathic helical region of human SP-B, efficiently mimics some functional properties of SP-B and is included in therapeutic surfactant preparations used in trials to treat respiratory distress syndrome. The membrane orientation of this peptide is controversial. We used an in vitro transcription-translation system to study the insertion of hydrophob ...[more]

PMID: 18621816

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Project description:Hundreds of tail-anchored proteins, including soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) involved in vesicle fusion, are inserted post-translationally into the endoplasmic reticulum membrane by a dedicated protein-targeting pathway. Before insertion, the carboxy-terminal transmembrane domains of tail-anchored proteins are shielded in the cytosol by the conserved targeting factor Get3 (in yeast; TRC40 in mammals). The Get3 endoplasmic-reticulum receptor comprises the cytosolic domains of the Get1/2 (WRB/CAML) transmembrane complex, which interact individually with the targeting factor to drive a conformational change that enables substrate release and, as a consequence, insertion. Because tail-anchored protein insertion is not associated with significant translocation of hydrophilic protein sequences across the membrane, it remains possible that Get1/2 cytosolic domains are sufficient to place Get3 in proximity with the endoplasmic-reticulum lipid bilayer and permit spontaneous insertion to occur. Here we use cell reporters and biochemical reconstitution to define mutations in the Get1/2 transmembrane domain that disrupt tail-anchored protein insertion without interfering with Get1/2 cytosolic domain function. These mutations reveal a novel Get1/2 insertase function, in the absence of which substrates stay bound to Get3 despite their proximity to the lipid bilayer; as a consequence, the notion of spontaneous transmembrane domain insertion is a non sequitur. Instead, the Get1/2 transmembrane domain helps to release substrates from Get3 by capturing their transmembrane domains, and these transmembrane interactions define a bona fide pre-integrated intermediate along a facilitated route for tail-anchor entry into the lipid bilayer. Our work sheds light on the fundamental point of convergence between co-translational and post-translational endoplasmic-reticulum membrane protein targeting and insertion: a mechanism for reducing the ability of a targeting factor to shield its substrates enables substrate handover to a transmembrane-domain-docking site embedded in the endoplasmic-reticulum membrane.

Dataset Information

The surfactant peptide KL4 sequence is inserted with a transmembrane orientation into the endoplasmic reticulum membrane.

Publications

The surfactant peptide KL4 sequence is inserted with a transmembrane orientation into the endoplasmic reticulum membrane.

Similar Datasets

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