September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
The interaction of BFSP1 and its proteolytically derived fragments with cell membranes
Author Affiliations & Notes
  • Roy A Quinlan
    School of Biological and Biomedical Sciences, Biophysical Sciences Inst, Durham Univ, Durham, United Kingdom
    Biophysical Sciences Institute, UNiversity of Durham, Durham, United Kingdom
  • Antal Tapodi
    Department of Biochemistry and Medical Chemistry, Pecs, Hungary
  • Daniel Clemens
    Life Sciences Addition, UC Berkeley, Berkeley, California, United States
  • Karinne Nemeth-Cahalan
    Physiology and Biophysics, UC Irvine, Irvine, California, United States
  • Irene Vorontsova
    Physiology and Biophysics, UC Irvine, Irvine, California, United States
  • Peter Jakus
    Department of Biochemistry and Medical Chemistry, Pecs, Hungary
  • Chris Cawood
    School of Biological and Biomedical Sciences, Biophysical Sciences Inst, Durham Univ, Durham, United Kingdom
  • Miguel Jarrin
    School of Biological and Biomedical Sciences, Biophysical Sciences Inst, Durham Univ, Durham, United Kingdom
  • James Ewbank Hall
    Physiology and Biophysics, UC Irvine, Irvine, California, United States
  • Footnotes
    Commercial Relationships   Roy Quinlan, Fight for Sight UK (S); Antal Tapodi, None; Daniel Clemens, None; Karinne Nemeth-Cahalan, None; Irene Vorontsova, None; Peter Jakus, None; Chris Cawood, None; Miguel Jarrin, None; James Hall, None
  • Footnotes
    Support  Fight for Sight UK 1584/5, EY05661
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3074. doi:
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    • Get Citation

      Roy A Quinlan, Antal Tapodi, Daniel Clemens, Karinne Nemeth-Cahalan, Irene Vorontsova, Peter Jakus, Chris Cawood, Miguel Jarrin, James Ewbank Hall; The interaction of BFSP1 and its proteolytically derived fragments with cell membranes. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3074.

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

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Abstract

Purpose : To determine the mechanism of plasma membrane binding and function of BFSP1 in this cell compartment.

Methods : Lens membranes were prepared by sequential extraction, culminating with a treatment with 0.1M sodium hydroxide, a standard biochemical approach for the purification ofintegral membrane proteins. Full length human BFSP1 was obtained from Source Bioscience (http://www.lifesciences.sourcebioscience.com/ IMAGE clones 6154051 and 5406467). The C-terminal region of HsBFSP1 was amplified by PCR, the products cloned into and sequenced in pGEM-T Easy (Promega, UK; www.promega.com). These were inserted into the following vector systems: pET23/28 retaining the C-terminal His tag for subsequent purification; peGFPN3 for expression in tissue culture cells by transient transfection; pXβG for expression in Xenopus oocytes for oocyte swelling assays. BFSP1 and its fragments were produced recombinantly, purified and then used in biochemical assays. Transfected cells were imaged either using a confocal Leica SP5 laser scanning microscope or for live cell imaging experiments, an Andor Revolution XD Spinning Disc Confocal Laser Microscope system equipped with a live cell incubator system to regulate temperature and levels of carbon dioxide.

Results : BFSP1 fragments are found associated with the lens membrane fractions. BFSP1 is not an integral membrane protein but possesses several predicted amphipathic helices, one of which is coincident with a cryptic myristoylation sequence, which is revealed by caspase cleavage of BFSP1. Using epitope specific antibodies, we find the C-terminal sequences 434-665 are retained in the membrane fraction. These sequences contain 4 potential lipid binding domains. Transfection into MCF7 cells of various C-terminal sequences derived from the 434-665 identify one (434-460) capable of binding directly to membranes and capable of targeting GFP to this compartment. Functional assays (Xenopus oocyte) provide evidence for an indirect mechanism.

Conclusions : BFSP1 binds to the plasma membranes and internal membranes of cells. The 434-460 sequence is sufficient to partition a GFP tag to this compartment, but an indirect mechanism likely exists for the other BFSP1 sequences.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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