Ultrastructural localisation and further biochemical characterisation of prolyl 4-hydroxylase from Phaseolus vulgaris: comparative analysis

Prolyl 4-hydroxylase (EC 1.14,11.2), the enzyme responsible for the post-tr anslational hydroxylation of peptide proline, has been well described in an imals but has been little studied in plants. The best characterised example is the enzyme from French bean (Phaseolus vulgaris). In this study, the bi ochemical properties of this plant enzyme were examined in more detail and, using specific polyclonal antibodies, the localisation of the enzyme was d etermined. Both alpha- and beta-subunits did not show multiple forms; sugge sting a relatively broad specificity of the enzyme complex with respect to the target hydroxylated amino acid sequences. Antibodies to the mammalian a nd Chlamydomonas enzymes cross-react with the higher plant subunits, indica ting that some epitopes are highly conserved. The P. vulgaris enzyme was in hibited by analogues of oxoglutarate, but was not susceptible to doxorubici n. Inhibition of the bean enzyme by an oxaloglycine derivative resulted in the retention of the target (hydroxy)proline-rich protein in the endomembra ne system. Immunolocalisation of the enzyme showed close association with t he endoplasmic reticulum and Golgi apparatus in root tip cells of P. vulgar is or I Tropaeolum majus. This localisation was particularly pronounced in Golgi-associated vesicles of young root tip cells of T. majus, cell types w here rapid synthesis and deposition of wall material was observed. These da ta are consistent with the hypothesis, proposed by Bolwell [G.P. Bolwell, D ynamic aspects of the plant extracellular matrix, Int. Rev. Cytol. 146 (199 3) 261-324], that protein hydroxylation must be completed before the glycos ylation of the target (hydroxy)proline-rich glycoproteins in the Golgi stac k. (C) 1999 Elsevier Science Ltd. All rights reserved.