Signal sequence of human preproparathyroid hormone is inactive in yeast.
The biosynthesis of human preproparathyroid hormone (hpreproPTH) and the processing to mature parathyroid hormone (hPTH) was investigated in yeast. Cells were transformed with a plasmid that carried a fusion gene made of the yeast pyruvate kinase promoter, complementary DNA (cDNA) encoding a slightly modified form of hpreproPTH and the transcription termination signal from yeast triosephosphate-isomerase. In transformed yeast cells we identified a protein that was recognized by a PTH antiserum and, on gel electrophoresis, comigrated with hpreproPTH marker. The amino-terminal sequence of the protein was consistent with that of hpreproPTH, indicating that the hormone precursor is not processed. It was localized inside the cell, when analyzed in pulse-chase experiments by trypsin accessibility in intact and lysed spheroplasts. In contrast, when mRNA from these yeast cells and from human parathyroid tissue was translated into preproPTH in a reticulocyte lysate supplemented with canine pancreatic microsomes, the preproPTHs from both mRNAs were transported and cleaved with identical efficiencies. We conclude that hpreproPTH is synthesized in yeast but not recognized and processed like a precursor of a secreted protein by the yeast secretory apparatus.
Born
W
W
Endocrine Unit, Massachusetts General Hospital, Boston 02114.
Freeman
M
M
Bornstein
W
W
Rapoport
A
A
Klein
R D
RD
Hendy
G N
GN
Khorana
H G
HG
Rich
A
A
Potts
J T
JT
Kronenberg
H M
HM
D000818:Animals; D004285:Dogs; D010281:Parathyroid Hormone / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D015003:Yeasts / Q000378:metabolism*
D010281:Parathyroid Hormone; D011498:Protein Precursors; D021382:Protein Sorting Signals; C019056:preproparathormone