Structural features of the plant N‐recognin ClpS1 and sequence determinants in its targets that govern substrate selection

Abstract

In the N‐degron pathway of protein degradation of Escherichia coli, the N‐recognin ClpS identifies substrates bearing N‐terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N‐degron pathway. The residue in second position influences recognition (especially in N‐terminal ends starting with leucine). N‐terminal acetylation abrogates recognition. ClpF, a ClpS1‐interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate‐binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N‐degron pathway.

The CLP and PREP protease systems coordinate maturation and degradation of the chloroplast proteome in Arabidopsis thaliana

“…Furthermore, Nt tyrosine was shown in yeast and Arabidopsis mitochondria to be removed by ICP55 and to be a destabilizing residue (Vogtle et al ., 2011; Carrie et al ., 2015; Huang et al ., 2015). In chloroplasts, binding of CLPS1 to Nt tyrosine is restricted and depends on the residue in the second position, that is, it is enhanced for YP (Aguilar Lucero et al ., 2021) but was not detected for YR (R, Arginine; Montandon et al ., 2019) but the impact of a neighboring S residue (as in the case observed here) is not known.…”

“…A second possibility is that the pathway via which improperly processed Nt proteoforms are normally removed by the peptidase network, in a quality control‐like mechanism, is blocked in peptidase mutants. This seems feasible; however, only few of the aberrant Nt proteoforms have plastid primary N‐degrons, such as phenylalanine, tyrosine, or tryptophan (Apel et al ., 2010; Carrie et al ., 2015; Rampello & Glynn, 2017; Bouchnak & van Wijk, 2019, 2021; Aguilar Lucero et al ., 2021; Kim et al ., 2021). A third possibility is that the limited precision of SPP under conditions of imbalanced proteostasis, folding stress and peptide fragment accumulation causes interference between substrates and SPP (Fig.…”

Proteomics, phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP chaperone-protease system

“…We did evaluate for possible plastid N-degrons ( 5 , 68 ), and we observed three times a Leu (UVR3, HugZ-3, and DUF760-7) and once an Asp (UVR4) as the likely N-terminal residue. It was recently shown that N-terminal Leu is recognized by CLPS1 but that the following residue (the P2′ position) greatly affects the affinity, with Arg and also Gly enhancing the affinity but Leu, Ser, and Ala reducing affinity ( 68 ). Leu was followed by a Ser for HugZ-3 and DUF760-7 but Phe in case of UVR3.…”