期刊
EXPERIMENTAL EYE RESEARCH
卷 77, 期 4, 页码 505-514出版社
ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1016/S0014-4835(03)00151-9
关键词
peripherin/rds; membrane fusion; photoreceptors; GST-fusion protein
资金
- NCRR NIH HHS [S10 RR026365, S10 RR026365-01] Funding Source: Medline
- NEI NIH HHS [R01 EY010420-12, R21 EY018705-02, R01 EY010420, R01 EY010420-08, R01 EY010420-14, R01 EY010420-06, R01 EY010420-13A2, R29 EY010420, R01 EY010420-11, R21 EY018705-01A1, EY 03328, R21 EY018705, R01 EY010420-16, R01 EY010420-09, R01 EY010420-07, R01 EY013246, R29 EY010420-03, EY 13246, R01 EY010420-10A2, R01 EY010420-15, EY 10420] Funding Source: Medline
Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds. This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding. In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds. Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311-325) is essential for mediating lipid bilayer fusion of model membranes. To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284-346) were generated and polypeptides expressed. A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site. Protein expression was induced in E. coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS-PAGE and Western blot analysis. The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS-PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2136 (gift from Dr R. Molday). The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing. Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps. A common feature of most membrane fusion proteins is a change in conformation upon membrane association. Structural changes in the C-terminal polypeptide were investigated using far UV CD. The far UV CD spectra of the purified C-terminal polypeptide indicated substantial a-helical content in the wt peptide in isotonic aqueous buffer. An increase in intensity of 208 and 222 nm CID bands upon addition of DPC vesicles indicated an increase in a-helical content of the polypeptide. These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in a-helical content. (C) 2003 Elsevier Ltd. All rights reserved.
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