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William M. Dismuke, Brian S. McKay, W Daniel Stamer; Myocilin’s Coiled-Coil: pH-Dependent Protein Binding and Intracellular Targeting. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3674.
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© ARVO (1962-2015); The Authors (2016-present)
Myocilin is a widely expressed protein in which mutations result uniquely in ocular hypertension, glaucoma and blindness. We previously identified a SNARE-homology region in myocilin and showed that myocilin is a component of a membrane-associated protein complex. Here we test the hypothesis that the N-terminal, coiled-coil containing portion of myocilin (lacking the olfactomedin domain) is sufficient for targeting to the membrane-associated protein complex and examine conditions that mediate binding.
Membrane preparations from COS-7 cells stably expressing the N-terminal, coiled-coil-containing portion of myocilin (hth-CC-GFP) that lacks the olfactomedin domain were analyzed by velocity gradient sedimentation and western blot. A pull-down assay was developed using the coiled-coil portion of myocilin as bait for proteins from solubilized retina membrane preparation at two different pHs (7.4 and 5.5). Captured proteins were analyzed by SDS-PAGE and silver stain.
Fluorescent microscopy shows the hth-CC-GFP constructs associates with intracellular vesicles. Western blots show that membrane-associated hth-CC-GFP constructs separated on linear glycerol gradients (10-40%) into two distinct pools, a 8.6S fraction and a 16.6S fraction, similar to our previous findings with endogenous myocilin from human tissue. In our pull-down assay, the coiled-coil portion of myocilin associated with seven additional proteins at pH 5.5 compared to pH 7.4.
Our data demonstrates that the N-terminal portion of myocilin by itself is sufficient for targeting myocilin to a membrane-associated protein complex similar to the full-length protein. In this portion of myocilin we previously identified a region of the coiled-coil with homology to SNARE proteins. Myocilin's coiled-coil differs at a few key positions where acidic residues are present instead of neutral polar or hydrophobic residues. In low pH cellular compartments such as endosomes, vesicles, multivesicular bodies or Golgi Apparatus, these acidic residues become more neutral, possibly favoring protein-protein interactions consistent with our pull-down results.
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