title: Dual localization of wild-type myocilin in the endoplasmic reticulum and extracellular compartment likely occurs due to its incomplete secretion.
abstract: OBJECTIVE
  Wild-type myocilin is known to be secreted extracellularly, but a significant amount of the protein is also present in the endoplasmic reticulum (ER). The present study was undertaken to address whether intracellular myocilin is a true ER resident protein.
  
  
  METHODS
  Human wild-type myocilin was adenovirally expressed in human trabecular meshwork cells, and general characteristics of both intracellular and extracellular myocilins including molecular weight, pI, glycosylation state, and cleavage site of the signal peptide were examined by biochemical analyses. Topology and decay kinetics of myocilin were also examined by protease protection assay and pulse chase analysis, respectively. The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry.
  
  
  RESULTS
  Intracellular myocilin were very similar to secreted myocilin in characteristics such as molecular weight, pI, glycosylation state, and cleavage site of the signal peptide. The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus. The kinetics of myocilin turnover clearly showed that it was intrinsically a very stable but incompletely secreted protein. The expression of myocilin was confined to a subset of cells and accompanied by the upregulation of a 78 kDa glucose-regulated protein, suggesting that it was not properly folded or processed in the ER.
  
  
  CONCLUSIONS
  Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.
authors.affiliation: Department of Ophthalmology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea.
authors.forename: Seongsoo
authors.initials: S
authors.lastname: Sohn
authors.forename: Myung Kuk
authors.initials: MK
authors.lastname: Joe
authors.forename: Tae Eun
authors.initials: TE
authors.lastname: Kim
authors.forename: Ji-Eun
authors.initials: JE
authors.lastname: Im
authors.forename: Young Ran
authors.initials: YR
authors.lastname: Choi
authors.forename: Hwayong
authors.initials: H
authors.lastname: Park
authors.forename: Changwon
authors.initials: C
authors.lastname: Kee
mesh_terms: D002460:Cell Line; D049109:Cell Proliferation; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005110:Extracellular Space / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005902:Glaucoma, Open-Angle / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D006801:Humans; D018832:Molecular Chaperones / Q000096:biosynthesis; D009842:Oligopeptides; D010455:Peptides; D017510:Protein Folding; D011993:Recombinant Fusion Proteins; D055571:Secretory Pathway; D013997:Time Factors; D014129:Trabecular Meshwork / Q000378:metabolism; D015854:Up-Regulation; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D005136:Eye Proteins; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000099179:Myocilin; C085706:FLAG peptide