Small proteins as regulators of archaeal membrane transporters in Methanosarcina mazei

The aim of this work was the characterization and functional investigation of selected verified small proteins. Altogether four small proteins were characterized (sP36, MtrR, MttS and MttQ) and shown to be involved in the regulation of key membrane complexes. The 61 aa protein sP36, identified by mass spectrometry, was shown to be highly upregulated under nitrogen limitation on the transcript and the protein level. After increased ammonium concentration, sP36 facilitates the interaction of GlnK1 and AmtB1. This interaction results in the inactivation of AmtB1 through GlnK1, which decreases energy loss due to futile cycling of ammonium and prevents ammonium toxicity. MtrR is a 49 aa protein, demonstrated that MtrR is localized exclusively at the membrane. Using MtrR in a pulldown experiment targeting the M. mazei membrane fraction, two subunits of the tetrahydrosarcinapterin-S-methyltransferase (Mtr) were copurified. MtrR is required to effectively use H2 for methane production independent of the carbon source through modulation of the Mtr-reaction. Two further small proteins, MttQ (97 aa) and MttS (49 aa), are encoded in very close 3’-proximity of a gene encoding the putative transport protein MttP. Here, the three genes are demonstrated to be organized in one large operon including four other genes, which are all involved in the degradation of TMA and dimethylamine. MttP was proposed to be a TMA transporter, which was experimentally validated for the first time in this study as a complex with the small proteins MttQ and MttS. Further, a chromosomal deletion of mttS results in slower growth and lower maximum cell density in comparison to the wt with TMA as sole carbon source. This demonstrates that the small protein is a crucial subunit for the TMA transport complex.

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