A Single C-terminal Peptide Segment Mediates Both Membrane Association and Localization of Lysyl Hydroxylase in the Endoplasmic Reticulum

Abstract

Hydroxylation of lysyl residues is crucial for the unique glycosylation pattern found in collagens and for the mechanical strength of fully assembled extracellular collagen fibers. Hydroxylation is catalyzed in the lumen of the endoplasmic reticulum (ER) by a specific enzyme, lysyl hydroxylase (LH). The absence of the known ERspecific retrieval motifs in its primary structure 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 down the transport of the reporter protein from the ER into post-ER compartments, as assessed by our pulse-chase experiments. The retardation efficiency mediated by this Cterminal peptide segment is comparable with that of the intact LH but lower than that of the KDEL receptorbased retrieval mechanism. 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 localization, because the last 15 C-terminal amino acids did not possess substantial retardation activity alone. Our findings thus define a short peptide segment very close to the extreme C terminus of LH as the only necessary determinant both for its membrane association and localization in the ER.

Dimerization of human lysyl hydroxylase 3 (LH3) is mediated by the amino acids 541–547

“…There is a high homology between the LH isoforms, the conserved amino acids are found primarily in the carboxy-terminal portion of the molecules. The LH isoforms are located in the ER, loosely bound to the endoplasmic membrane (Kellokumpu et al, 1994;Salo et al, 2006;Suokas et al, 2000). In addition, LH3 is also located in the extracellular space , which differentiates LH3 from the other isoforms.…”

Defective Acidification of Intracellular Organelles Results in Aberrant Secretion of Cathepsin D in Cancer Cells

“…Metabolic Labeling and Immunoprecipitations-Cells were metabolically labeled as described earlier (29). Briefly, cells were starved for 30 min in cysteine/methionine-free Dulbecco's modified Eagle's Medium (Sigma-Aldrich) supplemented with 1% fetal calf serum and antibiotics and then cultivated for 6 h to overnight in the presence of 200 Ci/ml Pro-Mix TM L- 35 S in vitro cell labeling mix (Amersham Biosciences).…”

The lysyl hydroxylase isoforms are widely expressed during mouse embryogenesis, but obtain tissue- and cell-specific patterns in the adult

“…This suggests that LH3 is needed in the biosynthesis of collagens in a fast growing embryo, and that its function in liver tissue changes after birth. Since all lysyl hydroxylase isoforms are highly homologous and lack a KDEL motif, an ER retention signal (Pelham, 1991), the retention of LH2 and LH3 in the ER and their association with ER membranes probably occurs via the mechanism described for LH1 (Kellokumpu et al, 1994;Suokas et al, 2000).…”