Jack J. Liang

Confocal fluorescence microscopy study of interaction between lens MIP26/AQP0 and crystallins in living cells

MIP26/AQP0 is the major lens fiber membrane protein and has been reported to interact with many other lens components including crystallins, lipid, and cytoskeletal proteins. Regarding crystallins, many previous reports indicate that MIP26/AQP0 interacts with either only α‐crystallin or some specific γ‐crystallins. Considering the possibly important role of MIP26/AQP0 in the reduction of light scattering in the lenses, we have further investigated its interaction with crystallins using confocal fluorescence resonance energy transfer (FRET) microscopy. Specifically, we used MIP26 tagged with a green fluorescence protein (GFP) as a donor and a crystallin (αA‐, αB‐, βB2‐, or γC‐crystallin) tagged with a red fluorescence protein (RFP) as an acceptor. The two plasmids were cotransfected to HeLa cells. After culture, laser scattering microscopy images were taken in each of the three channels: GFP, RFP, and FRET. The net FRET images were then obtained by removing the contribution of spectral bleed‐through. The pixels of net FRET were normalized with those of GFP. The results show the presence of measurable interactions between MIP26 and all crystallins, with the extent of interactions decreasing from αA‐ and αB‐crystallin to βB2‐ and γC‐crystallin. Competitive interaction study using untagged αA‐crystallin shows decreased net FRET, indicating specificity of the interactions between MIP26 and αA‐crystallin. We conclude that all crystallins interact with MIP26, the physiological significance of which may be a reduction in the difference of refractive index between membrane and cytoplasm. J. Cell. Biochem. 104: 51–58, 2008.

Oligomerization with wt αA- and αB-crystallins reduces proteasome-mediated degradation of C-terminally truncated αA-crystallin.

PURPOSE We previously demonstrated that the ubiquitin-proteasome pathway (UPP) is a general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminally truncated αA-crystallin. The objective of this work was to determine the effects of wt αA- and αB-crystallins on the degradation of C-terminally truncated αA-crystallin (αA(1-162)) and vice versa. METHODS Recombinant wt αA, αB, and αA(1-162) were expressed in Escherichia coli and purified to homogeneity by chromatography. Subunit exchange and oligomerization were detected by fluorescence resonance energy transfer (FRET), multiangle-light scattering and coprecipitation assays. Protein substrates were labeled with (125)I and lens epithelial cell lysates were used as the source of the UPP for degradation assays. RESULTS FRET, multiangle light scattering, and coprecipitation assays showed that αA(1-162) exchanged subunits with wt αA- or wt αB- crystallin to form hetero-oligomers. αA(1-162) was more susceptible than wt αA-crystallin to degradation by the UPP. When mixed with wt αA-crystallin at 1:1 or 1:4 (αA(1-162) : wt) ratios to form hetero-oligomers, the degradation of αA(1-162) was significantly decreased. Conversely, formation of hetero-oligomers with αA(1-162) enhanced the degradation of wt αA-crystallin. The presence of αA(1-162), but not wt αA-crystallin, decreased the degradation of wt αB-crystallin. CONCLUSIONS αA(1-162) forms hetero-oligomers with wt αA- and αB-crystallins. Oligomerization with wt αA- or αB-crystallins reduces the susceptibility of αA(1-162) to degradation by the UPP. In addition, the presence of αA(1-162) in the hetero-oligomers also affects the degradation of wt αA- and αB-crystallins.