Small subunits MttS and MttQ of the MttP transporter regulate trimethylamine transport in Methanosarcina mazei

Small proteins (<100 aa) have moved in the focus of science after being overlooked for decades due to bioinformatical and biochemical challenges. While mass spectrometry-coupled ribosome profiling of the mesophilic methanoarchaeon Methanosarcina mazei has recently unveiled a wealth of novel small ORFs, the functional roles of most of their products remain unknown. Here, we report the characterization of MttQ (98 aa) and MttS (49 aa), products of small ORFs situated in an operon alongside genes encoding a drug-metabolite-efflux (DME) family transporter (mttP) and other enzymes involved in trimethylamine (TMA) degradation. MttS and MttQ interact with MttP to form a stable oligomeric complex spanning the cytoplasmic membrane. TMA transport activity of the MttQ/MttS/MttP-complex is demonstrated via in vivo Escherichia coli cells heterologously expressing this system. Based on the reduced growth of a M. mazei mutant lacking mttS, on TMA as sole carbon source, we conclude that MttS governs the specificity of TMA transport. We posit that interactions between a DME transporter, e.g., MttP, and small proteins fueled evolution of the MttPQS complex and the advent of selective TMA uptake in methylotrophic methanoarchaea. These findings suggest an evolutionary mechanism on how small accessory proteins can alter conserved core functions in order to explore new ecological niches. Further, given that TMA levels in the human bloodstream influence cardiovascular disease risk but can be degraded by host-associated methanoarchaea containing a homolog of the MttPQS-complex, our findings present insight into an archaeal pathway with relevance to human health.