The LonB protease has a global impact on the proteome synthesis in the haloarchaeon Haloferax volcanii

PAGGI, R.A.; ALBAUM, S.; CERLETTI, M.; TROTSCHEL, C.; DE CASTRO, R.E; RAMALLO GUEVARA, C.; POETSCH, A.

San Miguel de Tucuman

Congreso; XII Congreso Argentino de Microbiologia General; 2017

SAMIGE

Energy-dependent proteolysis is a key process in cell physiology. ATP-dependent Lon proteases are conserved among the three Domains of Life and in Archaea it is unusually associated to the cytoplasmic membrane (LonB subfamily). We have previously shown that LonB is an essential protease in the haloarchaeon Haloferax volcanii, and that suboptimal amounts of this protease affect growth rate, cell shape and produce hyperpigmentation. To better understand the biological relevance of Lon in archaeal cells, the whole proteome turnover was examined in a H. volcanii conditional LonB mutant (HVLON3) under reduced (- trp) and physiological (+ trp) protease levels. Liquid chromatography coupled to tandem mass spectrometry combined with stable isotope labeling was applied for the identification and quantitation of membrane and cytosol proteins affected by the LonB protease. In a previous report we showed that presence of LonB affected the degradation of many proteins and several potential LonB substrates were identified (including phytoene synthase, key enzyme in the carotenoid biosynthesis pathway). In this work we focused on the effect of LonB on the overall proteome synthesis. A total of 225 proteins displayed differential synthesis rates depending on LonB expression. Proteins related to translation, amino acids, co-enzyme, nucleotide and energy metabolism showed a decrease in synthesis when LonB was induced while those involved in transcription, environmental information processing and lipid metabolism displayed an increase in synthesis under this condition. Several proteins that showed LonB-dependent synthesis correlated accordingly with changes in protein amounts observed in our previous work that compared the proteomes of the HVLON3 mutant vs the parental H26 strain by a quantitative MS approach. For instance, a membrane protein of unknown function (HVO_A0039) whose synthesis was arrested after LonB induction, increased by up to 320 fold in HVLON3 (reduced Lon levels). This observation was also supported by an RT-PCR assay, showing that while the specific HVO_A0039 transcript was almost undetectable in H26, its level dramatically increased in HVLON3. The whole proteome turnover analysis, performed for the first time in an archaeon, shows that the membrane LonB protease not only has a global impact on protein degradation but also on the proteome synthesis in the archaeon H. volcanii.