XB-ART-3290J Biol Chem September 17, 2004; 279 (38): 39958-67.
Uncoupling of photoreceptor peripherin/rds fusogenic activity from biosynthesis, subunit assembly, and targeting: a potential mechanism for pathogenic effects.
Inherited defects in the RDS gene cause a multiplicity of progressive retinal diseases in humans. The gene product, peripherin/rds (P/rds), is a member of the tetraspanin protein family required for normal vertebrate photoreceptor outer segment (OS) architecture. Although its molecular function remains uncertain, P/rds has been suggested to catalyze membrane fusion events required for the OS renewal process. This study investigates the importance of two charged residues within a predicted C-terminal helical region for protein biosynthesis, localization, and interaction with model membranes. Targeted mutagenesis was utilized to neutralize charges at Glu(321) and Lys(324) individually and in combination to generate three mutant variants. Studies were conducted on variants expressed as 1) full-length P/rds in COS-1 cells, 2) glutathione S-transferase fusion proteins in Escherichia coli, and 3) membrane-associated green fluorescent protein fusion proteins in transgenic Xenopus laevis. None of the mutations affected biosynthesis of full-length P/rds in COS-1 cells as assessed by Western blotting, sedimentation velocity, and immunofluorescence microscopy. Although all mutations reside within a recently identified localization signal, none altered the ability of this region to direct OS targeting in transgenic X. laevis retinas. In contrast, individual or simultaneous neutralization of the charged amino acids Glu(321) and Lys(324) abolished the ability of the C-terminal domain to promote model membrane fusion as assayed by lipid mixing. These results demonstrate that, although overlapping, C-terminal determinants responsible for OS targeting and fusogenicity are separable and that fusogenic activity has been uncoupled from other protein properties. The observation that subunit assembly and OS targeting can both proceed normally in the absence of fusogenic activity suggests that properly assembled and targeted yet functionally altered proteins could potentially generate pathogenic effects within the vertebrate photoreceptor.
PubMed ID: 15252042
PMC ID: PMC1360210
Article link: J Biol Chem
Grant support: EY014803 NEI NIH HHS , EY10420 NEI NIH HHS , EY13246 NEI NIH HHS , R01 EY013246-01 NEI NIH HHS , R01 EY013246-02 NEI NIH HHS , R01 EY013246-03 NEI NIH HHS , R01 EY013246-03S1 NEI NIH HHS , R01 EY010420-09 NEI NIH HHS , R01 EY010420-10A2 NEI NIH HHS , R01 EY010420-11 NEI NIH HHS , R01 EY010420-12 NEI NIH HHS , R01 EY010420-13A2 NEI NIH HHS , R01 EY010420-14 NEI NIH HHS , R01 EY010420-15 NEI NIH HHS , R01 EY010420-16 NEI NIH HHS , R01 DE022465 NIDCR NIH HHS, R01 EY010420 NEI NIH HHS , R01 EY010420-06 NEI NIH HHS , R01 EY010420-07 NEI NIH HHS , R01 EY010420-08 NEI NIH HHS , R21 EY018705 NEI NIH HHS , R21 EY018705-01A1 NEI NIH HHS , R21 EY018705-02 NEI NIH HHS , R29 EY010420 NEI NIH HHS , R29 EY010420-03 NEI NIH HHS , S10 RR026365 NCRR NIH HHS , S10 RR026365-01 NCRR NIH HHS , R01 EY013246 NEI NIH HHS , R24 EY014803 NEI NIH HHS
Genes referenced: prph prph2
GO Terms: photoreceptor cell outer segment organization
Disease Ontology terms: retinal disease