Thylakoid membranes from Arabidopsis LC-MSMS

The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps, with a single conserved -sandwich fold domain defining the family, the α-crystallin domain, whereas the N-terminal and C-terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non-metazoan sHsp expressed in the chloroplasts in green plants, which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat-stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic 15N-isotope labelling, of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat-stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered

小分子热激蛋白（small heat shock protein, sHsp）分子伴侣对细胞存活至关重要，可阻止在去稳定条件下部分解折叠的底物蛋白发生聚集。目前针对sHsps分子伴侣活性的多数研究，均基于有限的热敏性模式底物蛋白集合——这是因为其内源靶标往往尚未明确。sHsp家族成员间存在高度多样性：该家族以单一保守的β三明治折叠结构域（α-crystallin domain）为特征，而不同sHsp的N端与C端区域在长度和序列上差异显著，仅在直系同源物间保持保守。其内源靶标或许也会因sHsp类型、细胞区室、细胞类型及生物体的不同而存在差异。本研究针对Hsp21展开了探究——这是一种在绿色植物叶绿体中表达的非后生动物源sHsp，而叶绿体所处环境会经历剧烈的波动，尤以温度变化最为显著。我们阐明了Hsp21与叶绿体类囊体膜的相互作用机制：无论是在体外将分离的类囊体膜与Hsp21蛋白共孵育，还是在植物遭受热胁迫时，均能观察到二者的结合。通过对重组表达纯化的Hsp21蛋白，或是完整拟南芥（Arabidopsis thaliana）植株进行代谢性15N同位素标记（15N-isotope labelling）后，借助定量质谱（quantitative mass spectrometry）技术，我们精准测定了与类囊体膜结合的Hsp21含量。研究发现，在热胁迫植株中，仅有少数蛋白会与类囊体膜结合，而Hsp21正是其中之一；且叶绿体中约三分之二的Hsp21库会受到热胁迫的影响。我们由此得出结论：若要全面阐明sHsps在植物抗逆性中的作用，还需将其与膜结构的结合纳入考量范畴。