Abstract
The essential chloroplast CLP protease system consists of a tetradecameric proteolytic core with catalytic P (P1, 3–6) and non‐catalytic R (R1–4) subunits, CLP chaperones and adaptors. The chloroplast CLP complex has a total of ten catalytic sites,but it is not known how many of these catalytic sites can be inactivated before plants lose viability. Here we show that CLPP3 and the catalytically inactive variant CLPP3S164A fully complement the developmental arrest of the clpp3‐1 null mutant, even under environmental stress. In contrast, whereas the inactive variant CLPP5S193A assembled into the CLP core, it cannot rescue the embryo lethal phenotype of the clpp5‐1 null mutant. This shows that CLPP3 makes a unique structural contribution but its catalytic site is dispensable, whereas the catalytic activity of CLPP5 is essential. Mass spectrometry of affinity‐purified CLP cores of the complemented lines showed highly enriched CLP cores. Other chloroplast proteins were co‐purified with the CLP cores and are candidate substrates. A strong overlap of co‐purified proteins between the CLP core complexes with active and inactive subunits indicates that CLP cores with reduced number of catalytic sites do not over‐accumulate substrates, suggesting that the bottle‐neck for degradation is likely substrate recognition and unfolding by CLP adaptors and chaperones, upstream of the CLP core.
Highlights
ATP‐dependent Clp proteases are present in bacteria, as well as mitochondria and plastids, where they regulate accumulation levels of a broad range of substrates (Alexopoulos, Guarne, & Ortega, 2012; Liu, Ologbenla, & Houry, 2014; Nishimura, Kato, & Sakamoto, 2017; Nishimura & van Wijk, 2015; Sauer & Baker, 2011)
The catalytic triad Ser‐His‐Asp in CLPP proteases is widely conserved across bacterial CLPP proteins as well mitochondrial and plastid CLPP (Kim & Kim, 2008; Olinares et al, 2011; Peltier et al, 2004) (Supporting information Figure S1)
To determine if the contribution of CLPP3 to the core complex is through its unique structure or by contributing a catalytic site to the core complex, we transformed heterozygous clpp3-1 mutants with genomic CLPP3 with a C‐terminal STREPII tag (CLPP3-STREPII), or the same genomic construct but with a serine to alanine mutation in the catalytic site (CLPP3S164A-STREPII) rendering CLPP3 catalytically inactive
Summary
ATP‐dependent Clp proteases are present in bacteria, as well as mitochondria and plastids (organelles of bacterial origin), where they regulate accumulation levels of a broad range of substrates (Alexopoulos, Guarne, & Ortega, 2012; Liu, Ologbenla, & Houry, 2014; Nishimura, Kato, & Sakamoto, 2017; Nishimura & van Wijk, 2015; Sauer & Baker, 2011). To identify potential substrates using the CLP core trapping technique as explained in the INTRODUCTION, we carried out replicate affinity experiments for homozygous clpp3-1 complemented with CLPP3S164A-STREPII and using clpp3-1 complemented by CLPP3STREPII as a control.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
