Co-expression of the α subunit of human prolyl 4-hydroxylase with BiP polypeptide in insect cells leads to the formation of soluble and insoluble complexes. Soluble α-subunit-BiP complexes have no prolyl 4-hydroxylase activity

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Abstract
Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the post-translational formation of 4-hydroxyproline in collagens. The vertebrate enzymes are α2β2 tetramers, their β subunit being identical to protein disulphide isomerase (PDI). The function of the PDI-β subunit in prolyl 4-hydroxylases is not fully understood, but it seems to be that of keeping the highly insoluble α subunits in solution. We report here that expression of the α subunit of human type I prolyl 4-hydroxylase in insect cells together with BiP polypeptide leads to the formation of both soluble and insoluble α-subunit–BiP complexes. Formation of the soluble complexes was evident from (1) a marked increase in the amount of the α subunit in the soluble fraction of the cell homogenates when expressed together with BiP, (2) immunoprecipitation experiments and (3) demonstration of the presence of some of the complexes by polyacrylamide gel electrophoresis under non-denaturing conditions. Formation of the insoluble complexes was suggested by an increase in the amount of BiP in the insoluble fraction when expressed together with the α subunit. Nevertheless the soluble α-subunit–BiP complexes had no prolyl 4-hydroxylase activity. This indicates that the function of the PDI-β subunit in the prolyl 4-hydroxylase tetramer is not only that of keeping the α subunits in solution but appears to be more specific, probably that of keeping them in a catalytically active, non-aggregated conformation.