Structural determinants for signal sequence function in the mammalian endoplasmic reticulum.
Signal sequences function in protein targeting to and translocation across the endoplasmic reticulum membrane. To investigate the structural requirements for signal sequence function, chimeras of the Escherichia coli LamB signal peptide and prolactin were prepared. The LamB signal peptide was chosen by virtue of the extensive biophysical and biological characterization of its activity. In vitro, nascent prolactin chains bearing the LamB signal peptide (LamB) were targeted in a signal recognition particle (SRP)-dependent manner to rough microsomes but remained protease- and salt-sensitive and translocated at low efficiency. Full translocation activity was obtained in a gain of function mutant (LamB*) in which three hydrophobic residues in the LamB hydrophobic core were converted to leucine residues. Cross-linking studies demonstrated that the LamB* signal sequence displayed markedly enhanced interactions with SRP and integral membrane proteins. In contrast, chemically denatured LamB and LamB*-precursors bound with identical efficiencies and in a salt-resistant manner to rough microsomes, suggesting that during de novo synthesis the signal sequence of LamB-bearing precursors assumes a conformation refractory to translocation. These data indicate that a leucine-rich signal sequence is necessary for optimal interaction with SRP and suggest that SRP, by maintaining the signal sequence in a conformation suitable for membrane binding, performs a chaperone function.
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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Nicchitta
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D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D008322:Mammals; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000378:metabolism*; D015398:Signal Transduction
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C085044:LamB signal peptide, E coli; D021382:Protein Sorting Signals