Jan 5, 2002

Protein interactions with myocilin

Investigative Ophthalmology & Visual Science
Kelly Wentz-HunterBeatrice Y J T Yue


To identify factors that interact in vivo with myocilin, a glaucoma gene product. The yeast two-hybrid system with myocilin as the bait and a human skeletal muscle cDNA library as the prey was used to identify potential factors that interact with myocilin. Interactions were also examined in bovine trabecular meshwork (TM) cells through a mammalian two-hybrid system. Biochemical coimmunoprecipitation from both human TM cell lysate and in vitro translated proteins was also used to confirm results obtained from yeast analysis. Twenty positive clones isolated through yeast two-hybrid screening were deemed potential myocilin partners. Sequence analysis determined that two of them encoded for myocilin from amino acids 64 to 268. Myocilin was also found to interact with a component of the myosin motor protein, myosin regulatory light chain (RLC). The myocilin-myocilin and myocilin-RLC interactions revealed by the yeast system were further confirmed and demonstrated in cultured TM cells, by means of a mammalian two-hybrid system, and through biochemical coimmunoprecipitation, subcellular fractionation, immunofluorescence, and immunogold double labeling. These results indicate that myocilin can form homomultimers in vivo, independent of...Continue Reading

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Mentioned in this Paper

Immunofluorescence Assay
Yeast Two-Hybrid System Techniques
Amino Acids, I.V. solution additive
Motor protein
Leucine Zippers
Actomyosin Location
Bos taurus
Indirect Immunofluorescence

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