COEXPRESSION OF THE ALPHA-SUBUNIT OF HUMAN PROLYL 4-HYDROXYLASE WITH BIP POLYPEPTIDE IN INSECT CELLS LEADS TO THE FORMATION OF SOLUBLE AND INSOLUBLE COMPLEXES - SOLUBLE ALPHA-SUBUNIT-BIP COMPLEXES HAVE NO PROLYL 4-HYDROXYLASE ACTIVITY

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the post-translational f ormation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha 2 beta 2 tetramers, their beta subunit being identical to protei n disulphide isomerase (PDI). The function of the PDI-beta subunit in prolyl 4-hydroxylases is not fully understood, but it seems to be that of keeping the highly insoluble alpha subunits in solution. We report here that expression of the alpha subunit of human type I prolyl 4-hy droxylase in insect cells together with BiP polypeptide leads to the f ormation of both soluble and insoluble alpha-subunit-BiP complexes. Fo rmation of the soluble complexes was evident from (1) a marked increas e in the amount of the alpha subunit in the soluble fraction of the ce ll homogenates when expressed together with Rip, (2) immunoprecipitati on experiments and (3) demonstration of the presence of some of the co mplexes by polyacrylamide gel electrophoresis under non-denaturing con ditions. Formation of the insoluble complexes was suggested by an incr ease in the amount of BiP in the insoluble fraction when expressed tog ether with the a subunit. Nevertheless the soluble alpha-subunit-BiP c omplexes had no prolyl 4-hydroxylase activity. This indicates that the function of the PDI-beta subunit in the prolyl 4-hydroxylase tetramer is not only that of keeping the a subunits in solution but appears to be more specific, probably that of keeping them in a catalytically ac tive, non-aggregated conformation.