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Bhupesh Parise, Birgit Lorenz, Markus N. Preising; Overlapping Intracellular Localization of AIPL1 and CENPF upon Heterologous Expression of His-Tagged AIPL1 Constructs. Invest. Ophthalmol. Vis. Sci. 2012;53(14):762.
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The photoreceptor specific chaperone AIPL1 is one of 18 proteins whose genes are involved in the etiology of early-onset severe retinal dystrophies (EOSRD). AIPL1 has been shown to underlie a very severe form of EOSRD attributed to its chaperone function on NUB1 and PDE6A. PDE6A-associated retinal degeneration is much less severe in progression than EOSRD and a NUB1-associated retinal phenotype is unknown. To understand the severity of the AIPL1-associated phenotype we sought to identify other possible partners for AIPL1 that imply a more severe phenotype upon disrupted interaction. Upon screening a photoreceptor specific cDNA-library by Yeast-2 Hybrid assay we isolated a clone containing the C-terminus of centromere-specific protein F gene (CENPF). Here we will present the results of intracellular immunostaining of AIPL1 and CENPF to evaluate a possible interaction of both proteins.
Full length AIPL1 and previously identified splice variants were cloned into pQE-Tris system (Qiagen) which contained a C-terminal His-tag. HeLa cells were seeded in silicone frames on microscope slides at 37°C and 5% CO2 over night in DMEM with 10% FCS and penicillin/streptomycin. The constructs were transfected and expressed in HeLa cells. Transfected HeLa cells were probed for AIPL1 by Anti-His-tag antibody (ab) or anti-AIPL1 ab against intrinsic CENPF. Immunoreactivity (IR) was recorded by epifluorescence.
IR against the anti-AIPL1 ab and the anti-His-tag ab for expression of the AIPL1 wildtype construct and its splice variants were seen in the cytoplasm of HeLa cells. IR to intrinsically expressed CENPF also localized to the cytoplasm in HeLa cells overlapping with the IR of AIPL1 and its splice variants.
AIPL1 has chaperone function on a set of proteins which has not yet been completely elucidated. An Y2H assay indicated an interaction of AIPL1 and CENPF. IR co-localization inside HeLa cells further supports this finding. Whether AIPL1 supports CENPF function or CENPF is needed for AIPL1 function is unclear at this time. With CENPF a partner in the housekeeping pathways of the cell is identified that would predict a severe and highly progressive phenotype seen in AIPL1 patients by its activity as microtubule organizer. Co-immunoprecipitation studies of AIPL1 and CENPF expressed in HEK293 cells are in progress.
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