Abstract
A functional analogue of the eukaryotic ubiquitin‐proteasome system (UPS) is found in archaea. This archaeal system, termed the SAMP‐proteasome system (SPS), operates in targeted protein turnover; however, many of its protein components [E1‐like UbaA, Cdc48/p97‐/Rpt‐ type AAA ATPases, and JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homologs] remain to be fully characterized. To dissect the role of the JAMM metalloprotease homologs within the SPS, we previously expressed and purified the two Haloferax volcanii JAMM/MPN+ homologs (HvJAMM1/2) from recombinant Escherichia coli. Only HvJAMM1 was demonstrated to be active as an Ubl isopeptidase. By contrast, HvJAMM2 was found to be required in vivo for protein turnover by the SPS. Here we focused on identifying and characterizing the protein partners and/or biochemical conditions which may be required in vitro for HvJAMM2 activity. For this objective, His6‐HvJAMM2 was expressed in H. volcanii and purified by Ni2+‐ chromatography to overcome factors that may be absent from recombinant E. coli. Co‐purifying protein partners were identified by LC‐MS/MS analysis, including HVO_2955 which is predicted to be a structural homolog of the JAMM/MPN− interacting RNA helicase Brr2 of the eukaryal spliceosome. While hvo_2955 was not essential for growth, we found an Δhvo_2955 mutant to have decreased growth under standard conditions, with the genetic defect trans complemented by expression of His6‐HVO_2955. Current work is focused on determining the role of HVO_2955 during stress as well as its potential binding to JAMM‐domain proteins in archaea. Assessing the role of this Brr2 structural homolog in RNA processing in conjunction with SPS components such as HvJAMM2 is anticipated to show a clearer picture of archaeal physiology.Support or Funding InformationFunds for this project were awarded to JM‐F through the Bilateral NSF/BIO‐BBSRC program (NSF 1642283), the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DEFG02‐05ER15650) and the National Institutes of Health (NIH R01 GM57498). Additional support is provided by the University of Florida's Preeminence Fellowship awarded to CR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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