Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase.

The correct folding and assembly of proteins within the endoplasmic reticulum (ER) are prerequisites for subsequent transport from this organelle to the Golgi apparatus. The mechanisms underlying the ability of the cell to recognize and retain unassembled or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the biosynthesis of procollagen. Here partially folded intermediates are retained and prevented from secretion, leading to a build up of unfolded chains within the cell. The accumulation of these partially folded intermediates occurs during vitamin C deficiency due to incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show that this retention is tightly regulated with little or no secretion occurring under conditions preventing proline hydroxylation. We studied the molecular mechanism underlying retention by determining which proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to procollagen during its biosynthesis is prolyl 4-hydroxylase, and no binding to other ER resident proteins including Hsp47 was detected. This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can recognize and retain unfolded procollagen chains and can release these chains for further transport once they have folded correctly.

A cautionary note when using pepsin as a probe for the formation of a collagen triple helix.

The ability of the collagen triple helix to resist digestion with proteases has been used as a conformational probe to ascertain whether a collagenous molecule has formed a correctly aligned helix during biosynthesis or during refolding of the protein in vitro. During our studies into the synthesis and folding of a variety of engineered procollagen polypeptide chains, we noted that resistance to digestion with proteases, in particular pepsin, could be misleading and does not necessarily indicate the formation of a collagen triple helix. These results clearly show that resistance to pepsin digestion alone should not be used to indicate correct folding and that preferably an alternative assay should be used to unequivocally demonstrate the formation of a correctly aligned triple helix.