A single C-terminal peptide segment mediates both membrane association andlocalization of lysyl hydroxylase in the endoplasmic reticulum

Hydroxylation of lysyl residues is crucial for the unique glycosylation pat tern found in collagens and for the mechanical strength of fully assembled extracellular collagen fibers. Hydroxylation is catalyzed in the lumen of t he endoplasmic reticulum (ER) by a specific enzyme, lysyl hydroxylase (LH). The absence of the known ER-specific retrieval motifs in its primary struc ture and its association with the ER membranes in vivo have suggested that the enzyme is localized in the ER via a novel retention/retrieval mechanism . We have identified here a 40-amino acid C-terminal peptide segment of LH that is able to convert cathepsin D, normally a soluble lysosomal protease, into a membrane-associated protein. The same segment also markedly slows d own the transport of the reporter protein from the ER into post-ER compartm ents, as assessed by our pulse-chase experiments. The retardation efficienc y mediated by this C-terminal peptide segment is comparable with that of th e intact LH but lower than that of the KDEL receptor-based retrieval mechan ism. Within this 40-amino acid segment, the first 25 amino acids appear to be the most crucial ones in terms of membrane association and ER localizati on, because the last 15 C-terminal amino acids did not possess substantial retardation activity alone. Our findings thus define a short peptide segmen t very close to the extreme C terminus of LH as the only necessary determin ant both for its membrane association and localization in the ER.