Abstract
The length of the hydrophobic core of the bovine parathyroid hormone signal peptide was modified by in vitro mutagenesis. Extension of the hydrophobic core by three amino acids at the NH2-terminal end had little effect on the proteolytic processing of the signal peptide by microsomal membranes. Deletion of 6 of the 12 amino acids in the core eliminated translocation and processing of the modified protein. Deletion of pairs of amino acids across the core resulted in position-dependent inhibition of signal activity unrelated to hydrophobicity but inversely related to the hydrophobic moments of the modified cores. Deletions in the NH2-terminal region of the core were strongly inhibitory for proteolytic processing whereas deletions in the COOH-terminal region had no effect or increased processing when assessed either co-translationally with microsomal membranes or post-translationally with purified hen oviduct signal peptidase. Deletion of cysteine 18 and alanine 19 increased processing, but deletion of cysteine alone or substitution of leucine for cysteine did not increase processing more than deletion of both residues at 18 and 19. Translations of the translocation-defective mutants with pairs of amino acids deleted in a wheat germ system were inhibited by addition of exogenous signal recognition particle suggesting that interactions of the modified signal peptides with signal recognition particle were normal. The position-dependent effects of the hydrophobic core modifications indicate that structural properties of the core in addition to hydrophobicity are important for signal activity. The parallel effects of the modifications on co-translational translocation and post-translational processing by purified signal peptidase suggest that proteins in the signal peptidase complex might be part of, or intimately associated with, membrane proteins involved in the translocation. A model is proposed in which the NH2-terminal region of the hydrophobic core binds to one subunit of the signal peptidase while the other subunit catalyzes the cleavage.
Highlights
The length of the hydrophobic core of the bovine The initial steps in the secretory pathway adrescribed by parathyroid hormone signal peptide was modified by the signal hypothesis (Blobel and Sabatini, 1971; Blobel and in vitro mutagenesis.Extension of the hydrophobic Dobberstein, 1975, Walter et al, 1984)
The parallel effects of the modifications on co-translationaltranslocationandpost-translational processing by purified signal peptidase suggest that proteins in the signal peptidasecomplex might bepart of, or intimately associated with, membrane proteins involved in translocationA. model is proposed in which the NHz-terminal regioonf the hydrophobic core binds ability of randomly chosen peptide sequences to function as signals when substituted for the natural signal peptides of yeast(Kaiser et al, 1987) or Escherichia coli (Bakerand Schatz, 1987) proteins suggests that their functionalrequirements are not stringent
We have shown that effects of deletions of amino acids within the hydrophobic core of the PTH signal peptide on bothco-translationaltranslocation and post-translational proteolytic processing are dependent on the position of the deletion
Summary
Biosystems (Foster City, CA) model 380A DNA synthesizer and purified by high performance liquid chromatography. T o examine whether increasing the length of the hydrophobic core would increase signal activity, a methionine form of prepro-PTH termed [19-Ala]prepro-PNTH (Mead et al, was substituted forlysine at position 9 (s9Met) effectively. Sequence eliminated premature termination of transcription by T7 RNA polymerase and did not affect processing of prepro-PNTH by microsomal membranes(Mead et al, 1986). This substitution mutation had little effect on translocation and processing to pro-PNTH(Fig. 1B). In contrast,processinsertion of 39 amino acids and is endo-Pro’”-Met’8b-G1y1k-Met1ffl- ing of the signal peptide toproduce pro-PNTH was abolished [19-52]PTH’8e-Met1ef-PTH(Mead et al, 1985). Oligonucleotide-directed Mutagenesis-The uracil-containing single-stranded DNA template form of pNT12 was propagated in E. coli CJ236 (Kunkel et al, 1987) with the interference resistance phage resulting in the protectioonf pro-PNTH processed from fulllength [19-Ala]prepro-PNTH( WT, Fig. lA),the unprocessed d10-15 protein was not protected from proteolyticdigestion indicatingthatthe modified protein was nottranslocated. Inactivity of this mutant signal peptide in which only six HydPreopptShidiogebniacl
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
