| Popis: |
The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is an ion transporter that creates and maintains intracellular calcium stores. SERCA is inhibited or stimulated by several membrane micropeptides including another-regulin (ALN), dwarf open reading frame (DWORF), endoregulin (ELN), phospholamban (PLB), and sarcolipin (SLN). We previously showed these micropeptides assemble into homo-oligomeric complexes with varying affinity. Here we tested whether different micropeptides can interact with each other, hypothesizing that co-assembly into hetero-oligomers may affect micropeptide bioavailability to regulate SERCA. We quantified the relative binding affinity of each combination of candidates using automated fluorescence resonance energy transfer (FRET) microscopy. All pairs were capable of interacting with good affinity, similar to that of to self-binding (homo-oligomerization). Testing each pair at a 1:5 ratio and a reciprocal 5:1 ratio, we noted that the affinity of hetero-oligomerization of some micropeptides depended on whether they were the minority or majority species. In particular, SLN was able to join oligomers when it was the minority species, but did not readily accommodate other micropeptides in the reciprocal experiment when it was expressed in 5-fold excess. The opposite was observed for ELN. PLB was a universal partner for all other micropeptides tested, forming avid hetero-oligomers whether it was the minority or majority species. Increasing expression of SERCA decreased PLB-DWORF hetero-oligomerization, suggesting that SERCA-micropeptide interactions compete with micropeptide-micropeptide interactions. Thus, micropeptides populate a regulatory network of diverse protein assemblies. The data suggest that the complexity of this interactome increases exponentially with the number of micropeptides that are coexpressed in a particular tissue. |
