Lysyl hydroxylase, a collagen processing enzyme, exemplifies a novel class of luminally-oriented peripheral membrane proteins in the endoplasmic reticulum.
Lysyl hydroxylase (LH), an enzyme required early during collagen biosynthesis, appears to be exceptional among proteins that are thought to be residents of the endoplasmic reticulum (ER). It is a homodimer and does not contain either of the two previously characterized ER-specific retention motifs (KDEL or the double lysine motif) in its primary structure. We now show that LH, nevertheless, resides in the lumen of the ER. In immunofluorescence experiments, LH co-localizes with a KDEL-containing protein, protein disulfide isomerase (PDI), and also co-sediments with it after fractionation of subcellular organelles by sucrose density gradient centrifugation. In addition, LH seems to be stress-inducible. In one respect, however, LH differs from PDI and other known luminal proteins in the organelle. It is found in situ only in association with the ER membranes. Our cell fractionation and Triton X-114 phase separation experiments suggest that it binds to the membranes via weak electrostatic interactions. LH can thus be regarded as a first luminally-oriented "peripheral membrane" protein which has been characterized in the ER. The results suggest a novel possibility by which ER lumen can acquire its specific protein components from the bulk flow.
Kellokumpu
S
S
Department of Anatomy, University of Oulu, Finland.
Sormunen
R
R
Heikkinen
J
J
Myllylä
R
R
D000595:Amino Acid Sequence; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D002501:Centrifugation, Zonal; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D007535:Isomerases / Q000032:analysis; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011189:Potassium Chloride / Q000494:pharmacology; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases; D012867:Skin / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology; D014415:Tunicamycin / Q000494:pharmacology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010455:Peptides; D014415:Tunicamycin; D011189:Potassium Chloride; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases