Substrates of the chloroplast small heat shock proteins 22E/F point to thermolability as a regulative switch for heat acclimation in Chlamydomonas reinhardtii

Abstract

Key messageWe have identified 39 proteins that interact directly or indirectly with high confidence with chloroplast HSP22E/F under heat stress thus revealing chloroplast processes affected by heat.AbstractUnder conditions promoting protein unfolding, small heat shock proteins (sHsps) prevent the irreversible aggregation of unfolding proteins by integrating into forming aggregates. Aggregates containing sHsps facilitate the access of Hsp70 and ClpB/Hsp104 chaperones, which in ATP-dependent reactions disentangle individual proteins from the aggregates and assist in their refolding to the native state. Chlamydomonas reinhardtii encodes eight different sHsps (HSP22A to H). The goal of this work was to identify chloroplast-targeted sHsps in Chlamydomonas and to obtain a comprehensive list of the substrates with which they interact during heat stress in order to understand which chloroplast processes are disturbed under heat stress. We show that HSP22E and HSP22F are major chloroplast-targeted sHsps that have emerged from a recent gene duplication event resulting from the ongoing diversification of sHsps in the Volvocales. HSP22E/F strongly accumulate during heat stress and form high molecular mass complexes. Using differential immunoprecipitation, mass spectrometry and a stringent filtering algorithm we identified 39 proteins that with high-confidence interact directly or indirectly with HSP22E/F under heat stress. We propose that the apparent thermolability of several of these proteins might be a desired trait as part of a mechanism enabling Chlamydomonas chloroplasts to rapidly react to thermal stress.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-017-0672-y) contains supplementary material, which is available to authorized users.

VIPP2 interacts with VIPP1 and HSP22E/F at chloroplast membranes and modulates a retrograde signal for HSP22E/F gene expression

“…Immunodetection was performed by enhanced chemiluminescence (ECL) using the FUSION‐FX7 Advance™ imaging system (PeqLab). Antisera were against VIPP1 (Liu et al, ), VIPP2 (this study), HSP70B and CGE1 (Schroda, Vallon, Whitelegge, Beck, & Wollman, ), CF1β (Lemaire & Wollman, ), LHCA2 (Agrisera AS01 006), PsbA (Agrisera AS05 084), SECA (Schroda M., unpublished), HSP22F (Rütgers et al, ), DEG1C (Theis, Lang, et al, ), HA (Abcam ab137838), PsaA (Agrisera AS06 172), cytochrome f (Pierre & Popot, ), HSP90C (Willmund & Schroda, ), and LHCSR3 (Naumann et al, ). Anti‐rabbit‐HRP (Sigma‐Aldrich) was used as secondary antibody.…”

“…Only three of the 125 proteins fulfilled these criteria, which were VIPP2 itself, VIPP1, and HSP22E/F. We considered HSP22E and HSP22F as a single entity because the mature proteins lacking chloroplast transit peptides differ by only eight amino acids (Rütgers et al, ), and thus mass spectrometry could not distinguish between the two proteins. All three proteins were detected in all experiments with a total of 4–19 unique peptides (Table ; Table S2; Figure S5).…”

“…Hsp21 has an N‐terminal amphipathic α‐helix harbouring six methionines that can get oxidized by H 2 O 2 and re‐reduced by a plastid PMSR (Gustavsson et al, ; Harndahl et al, ), suggesting an Hsp21 methionine sulfoxidation‐reduction cycle to quench reactive oxygen species (Sundby, Harndahl, Gustavsson, Ahrman, & Murphy, ). A similar mechanism has also been proposed for the membrane‐localized Hsp16.3 in Mycobacterium tuberculosis (Abulimiti, Qiu, Chen, Liu, & Chang, ). We have shown previously that HSP22E/F binds thermolabile soluble chloroplast proteins as well as chaperones HSP70B and CPN60B, most likely to prevent the formation of larger aggregates and to provide access for chaperones and proteases in order to refold and degrade aggregated proteins (Rütgers et al, ). Under conditions of oxidative stress, small HSPs have been shown to bind soluble enzymes and protect them from oxidative damage (Kitagawa, Miyakawa, Matsumura, & Tsuchido, ; Preville et al, ).…”

“…Mass spectrometry data were analysed with the MaxQuant software v1.6.0.1 (Cox et al, ; Cox & Mann, ) using default parameters and label‐free quantification (LFQ). The C. reinhardtii protein database consisted of the JGI v5.5 genome assembly and annotation including mitochondrial and plastid proteins (Rütgers et al, ).…”

A Musashi Splice Variant and Its Interaction Partners Influence Temperature Acclimation in Chlamydomonas

“…We have used a molecular approach to understand how microalgae react to changes in ambient temperature by identifying molecular components involved in the integration of temperature information in the model alga C. reinhardtii. These components influence the temperature acclimation (12-h temperature shifts during the dark period) of the microalgae, thus being linked directly to the survival rates under changing temperatures ranging from physiological (18°C, 28°C, and 37°C) to heat shock conditions (42°C) that start around 39°C for C. reinhardtii (Tanaka et al, 2000;Rütgers et al, 2017). These components include the three-RRM domaincontaining RNA-binding proteins C3 and Musashi variant 12, as well as XRN1, highlighting the importance of RNA metabolism for thermal acclimation in C. reinhardtii.…”