Prolyl 4-hydroxylase (P4-H) consists of two distinct polypeptides; the catalytically more important α-subunit and the β-subunit, which is identical to the multifunctional enzyme protein disulphide isomerase. The enzyme appears to be assembled in vivo into an α2β2 tetramer from newly synthesized α-subunits associating with an endogenous pool of β-subunits. Using a cell-free system, we have shown previously that enzyme assembly is redox-dependent and that assembled α-subunits are intramolecularly disulphide-bonded [John and Bulleid (1994) Biochemistry 33, 14018–14025]. Here we have studied this assembly process within intact cells by expressing both subunits in COS-1 cells. Newly synthesized α-subunits were shown to assemble with the β-subunit, to form insoluble aggregates, or to remain soluble but not associate with the β-subunit. Treatment of cells with dithiothreitol (DTT) led to dissociation of P4-H into subunits and on removal of DTT the enzyme reassembled. This reassembly was ATP-dependent, suggesting an interaction with an ATP-dependent chaperone. This was confirmed when immunoglobulin-heavy-chain binding protein (BiP) and α-subunits were co-immunoprecipitated with antibodies against the α-subunit and BiP, respectively. These results indicate that unassembled α-subunits are maintained in an assembly-competent form by interacting with the molecular chaperone BiP.
Hsc70, immunoglobulin heavy chain binding protein, and Hsp90 differ in their ability to stimulate transport of precursor proteins into mammalian microsomes.