Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum.
The trans-Golgi network (TGN) proprotein convertase furin is synthesized in a zymogenic form and is activated by intramolecular, autoproteolytic cleavage of the propeptide from its precursor. To obtain insight in possible functions of the furin propeptide, we have studied biosynthesis, propeptide cleavage, biological activity, and intracellular localization of human and bovine furin. Analysis of autocatalytic cleavage site mutants of furin revealed that efficient propeptide cleavage requires the presence of the complete furin cleavage consensus sequence Arg-X-Lys-Arg. In studies of a mutant in which the P1 + P4 + P5 residues of the autoproteolytic cleavage site were substituted, no substrate processing activity could be demonstrated, indicating a complete block of maturation. In immunofluorescence analysis, this mutant was found in the endoplasmic reticulum (ER), suggesting ER retention of profurin. This ER retention, however, appeared saturable. Furin proteins encoded by oxyanion hole mutant N188A and negative side chain mutant D248L, which possess autoprocessing activity but lack substrate processing activity, were found in the Golgi and the ER, respectively. Finally, analysis of a furin mutant, in which all three potential sites for N-linked glycosylation were altered, revealed autocatalytic cleavage, substrate processing, and transport to the Golgi. Our results indicate that cleavage of the propeptide occurs in the endoplasmic reticulum and is necessary but not sufficient for transport of furin out of this compartment.
Laboratory for Molecular Oncology, University of Leuven, Belgium.
J W
JW
Creemers
M
M
Vey
W
W
Schäfer
T A
TA
Ayoubi
A J
AJ
Roebroek
H D
HD
Klenk
W
W
Garten
W J
WJ
Van de Ven
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002417:Cattle; D002460:Cell Line; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D045683:Furin; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013381:Subtilisins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013552:Swine
D017931:DNA Primers; D010455:Peptides; D011498:Protein Precursors; D013381:Subtilisins; D045683:Furin