June 2020
Volume 61, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2020
Protein Interactions and Subcellular Localization of Lens Aquaporin-5
Author Affiliations & Notes
  • Kevin Schey
    Vanderbilt University, Nashville, Tennessee, United States
  • Romell Gletten
    Vanderbilt University, Nashville, Tennessee, United States
  • Footnotes
    Commercial Relationships   Kevin Schey, None; Romell Gletten, None
  • Footnotes
    Support  NIH Grant EY013462
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3475. doi:
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      Kevin Schey, Romell Gletten; Protein Interactions and Subcellular Localization of Lens Aquaporin-5. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3475.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Ocular lens aquaporin-5 (AQP5) is the second most abundant aquaporin in lens fiber cells and has been shown to move from cytoplasmic localization to plasma membrane localization from outer cortical cells to nuclear cells, respectively. The exact function of AQP5 in lens fiber cells remains unknown. The purpose of this work is to identify AQP5 interacting proteins and to determine subcellular localization of AQP5 to gain an understanding of AQP5 regulation and function in the lens.

Methods : Co-immunoprecipitation (coIP) of AQP5 and its interacting partner proteins with AQP5 antibodies was carried out on membrane preparations from bovine lenses dissected into cortical and nuclear regions. LC-MS/MS analysis was done to determine the identities and abundances of proteins that immunoprecipitated with AQP5. Significance Analysis of INTeractome (SAINT) software was used to determine significant interactions with AQP5. In addition, high resolution immunofluorescence (IF) confocal microscopy and electron microscopy (EM) analyses were carried out on bovine lens sections cut from fixed tissue. Both axial and equatorial orientations were examined. Airyscan confocal microscopy was used to provide high spatial resolution IF images.

Results : Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 (RPN2), proteasome subunit beta type-5, poly(rC)-binding protein 2, 14-3-3 protein eta, and ankyrin 3 were identified as potential cortical fiber cell AQP5 interacting partners in AQP5 coIP experiments.
IF microscopy showed punctate, spherical, and linear structures containing AQP5 in the outer cortical fiber cells near the equatorial bow region. EM analysis showed similar structures appearing to contain mitochondrial and endoplasmic reticulum components. Additional microscopy with mitochondrial markers and vimentin antibodies showed similar localization. Inner cortical and nuclear AQP5 was localized to the plasma membrane.

Conclusions : AQP5 is localized to mitochondrial and ER structures in the cytoplasm of outer cortical fiber cells before trafficking to the plasma membrane in older fiber cells. Cortical AQP5 appears to interact with multiple proteins associated with proteostasis and may serve a role in autophagy in lens fiber cells.

This is a 2020 ARVO Annual Meeting abstract.

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