Y-Complex Architecture Revisited: Super-Resolution Microscopy to Investigate Differences in Membrane Localization for...

Introduction: A key factor for the survival of an organism in its habitat is its ability to quickly adapt to changes in its environment on the protein level. One fast and efficient mechanism to influence protein abundance is the regulation of mRNA stability by ribonucleases. In the prokaryotic model organism Bacillus subtilis, the membrane-anchored RNase Y performs a crucial regulatory role by degrading and maturating mRNA. Previous studies have shown that RNase Y acts in concert with three proteins forming the Y-complex. In addition to its role as specificity factor for RNase Y, ribonuclease-independent functions have been proposed for the Y-complex during natural competence, biofilm formation, as well as sporulation.

In previous work, using single-molecule tracking, we showed that the Y-complex is highly dynamic and present in multiple compositions in vivo. Using biochemical analysis, recruitment of the Y-complex to RNase Y was shown to be mediated by YaaT, whereas YlbF and YmcA did not display any direct interactions. Here we employ 3D-structured illumination microscopy (SIM) super resolution and bimolecular fluorescence complementation (BiFC) to further characterize RNase Y/Y-complex localizations and interactions.

By visualizing the Y-complex proteins and RNase Y using SIM, we provide additional evidence that YaaT localizes differently than YmcA and YlbF, in that the fraction of YaaT is localized membrane-proximal appears to be higher than the one observed for YmcA and YlbF. We also show that the strength of YaaT membrane association is culture medium dependent. Evidence for membrane-proximal interaction of the Y-complex proteins with RNase Y is provided through the use of BiFC.

Taken together, our data support a model where the Y-complex is exclusively tethered to RNase Y by YaaT and where the composition of the Y-complex is fluctuating potentially in a function-dependent manner.