Characterization of rice small heat shock proteins targeted to different cellular organelles

Abstract

Small heat shock proteins (sHSPs) are a family of ATP-independent molecular chaperones which prevent cellular protein aggregation by binding to misfolded proteins. sHSPs form large oligomers that undergo drastic rearrangement/dissociation in order to execute their chaperone activity in protecting substrates from stress. Substrate-binding sites on sHSPs have been predominantly mapped on their intrinsically disordered N-terminal arms. This region is highly variable in sequence and length across species, and has been implicated in both oligomer formation and in mediating chaperone activity. Here, we present our results on the functional and structural characterization of five sHSPs in rice, each differing in their subcellular localisation, viz., cytoplasm, nucleus, chloroplast, mitochondria and peroxisome. We performed activity assays and dynamic light scattering studies to highlight differences in the chaperone activity and quaternary assembly of sHSPs targeted to various organelles. By cloning constructs that differ in the length and sequence of the tag in the N-terminal region, we have probed the sensitivity of sHSP oligomer assembly and chaperone activity to the length and amino acid composition of the N-terminus. In particular, we have shown that the incorporation of an N-terminal tag has significant consequences on sHSP quaternary structure.

Dodecameric structure of a small heat shock protein from Mycobacterium marinum M

“…It was interesting to observe that while deletion of residues from the N‐terminus (M1_ Δ N12) made the protein elute as a single population, the addition of residues at the N‐terminus (M1_His) made the protein a huge assembly, indicating direct involvement of N‐terminus in regulating the oligomeric size of the M1 particle. Similar observations were reported for sHSPs from Oryza sativa where addition of a hexa His‐tag toward the N‐terminus of these sHSPs altered their hydrodynamic radii and oligomeric states . M2 was extremely polydisperse and existed as a continuum of oligomers ranging from ~300 kDa to ~700 kDa (Supporting Information Figure S1D), while M3 had an oligomeric mass of 195.59 kDa (12‐mer) (Supporting Information Figure S1E).…”

“…Similar observations were reported for sHSPs from Oryza sativa where addition of a hexa Histag toward the N-terminus of these sHSPs altered their hydrodynamic radii and oligomeric states. 56 M2 was extremely polydisperse and existed as a continuum of oligomers ranging from~300 kDa tõ 700 kDa (Supporting Information Figure S1D), while M3 had an oligomeric mass of 195.59 kDa (12-mer) (Supporting Information Figure S1E).…”

Genome-wide identification of small heat-shock protein (HSP20) gene family in grape and expression profile during berry development

“…Under heat stress, HSP20s can prevent the aggregation and irreversible denaturation of heat-denatured proteins, which ensures that other proteins can perform normal functions at high temperature, providing a strong basis for improving the heat resistance of plant organs. HSP20s have been shown to be located in mitochondria, cytoplasm, and endoplasmic reticulum [16].…”

Genome-wide identification and classification of theHsfandsHspgene families inPrunus mume, and transcriptional analysis under heat stress

“…Most Hsp families (i.e., Hsp100, Hsp90, Hsp70, and Hsp60) are highly conserved across great organismal distance, and are among the most highly conserved protein families known (Stechmann & Cavalier-Smith, 2003; Waters, Aevermann & Sanders-Reed, 2008). Although the monomers of plant sHsp proteins are the smallest among Hsps (12–40 kDa), plant sHsps exhibit high diversity in amino acid sequence (Hilton et al, 2013; Mani, Ramakrishna & Suguna, 2015). Except for the conserved α-crystallin domain (ACD) near the C-terminus, the N- and C-terminal extensions are variable (Kriehuber et al, 2010; Haslbeck & Vierling, 2015).…”