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Biochemistry ; 46(5): 1256-72, 2007 Feb 06.
Article in English | MEDLINE | ID: mdl-17260955

ABSTRACT

Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/- retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2-ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST-PerCter protein selectively pulled down His-MREG and His-MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341-Gly346), and kinetic data predicted a KD of 80 nM for PerCter-MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk-plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function.


Subject(s)
Carrier Proteins/metabolism , Intermediate Filament Proteins/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Adaptor Proteins, Vesicular Transport , Animals , Binding Sites , Carrier Proteins/physiology , Cattle , Cell Line , Cell Membrane , Humans , Intermediate Filament Proteins/physiology , Intracellular Signaling Peptides and Proteins , Membrane Fusion , Membrane Glycoproteins/physiology , Membrane Proteins/metabolism , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/physiology , Optic Disk/ultrastructure , Peripherins , Photoreceptor Cells/ultrastructure , Rats , Retina/chemistry , Retina/cytology
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