Analysis of Lon1 protease traps to identify Arabidopsis protein substrates

ABSTRACT: The ATP-dependent Lon protease is a key component of protein quality control and highly conserved across the kingdoms of living organisms. In Arabidopsis thaliana Lon1 (At5g26860) is dual targeted to mitochondria and chloroplasts. Lon1 mutant plants (lon1-1) exhibit a post-embryonic growth retardation phenotype accompanied with aberrant mitochondria morphology and reduced respiratory capacity. Even though Lon degrades most of mitochondrial proteins, few substrates of Lon have been identified. To identify the Lon1 substrates, we generated transgenic Arabidopsis lon1-1 mutant plants introducing a proteolytically inactive variant of Lon1 (Lon1trap) targeted to mitochondria by mutating the Serine-Lysine catalytic dyad to Alanine residues. The Lon1trap bears a FLAG-tag at the C-terminus to immunoprecipitate the trap and collect the protein substrates of Lon1 protease immobilized in the proteolytic chamber. A Lon1wt variant that is proteolytically active and carries also a FLAG-tag was introduced to Arabidopsis lon1-1 mutant plants and used as a control. Total proteins were extracted under native conditions from both Lon1trap and Lon1wt transgenic plants of 8 days old seedling, grown vertically on petri dishes at 22oC in the dark from independent biological replicates. The proteins were incubated with anti-FLAG M2-agarose affinity gel to collect the Lon1 traps with the substrates by co-immunoprecipitation.

INSTRUMENT(S):

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Whole Body

SUBMITTER: Martina Samiotaki  

LAB HEAD: Stamatis Rigas

PROVIDER: PXD055807 | Pride | 2025-06-10

REPOSITORIES: Pride