Hsp78 (78 kDa Heat Shock Protein), a Representative AAA Family Member Found in the Mitochondrial Matrix of Saccharomyces cerevisiae

Abstract

ATPases associated with diverse cellular activities (AAA+) form a superfamily of proteins involved in a variety of functions and are characterized by the presence of an ATPase module containing two conserved motifs known as Walker A and Walker B. ClpB and Hsp104, chaperones that have disaggregase activities, are members of a subset of this superfamily, known as the AAA family, and are characterized by the presence of a second highly conserved motif, known as the second region of homology (SRH). Hsp104 and its homolog Hsp78 (78 kDa heat shock protein) are representatives of the Clp family in yeast. The structure and function of Hsp78 is reviewed and the possible existence of other homologs in metazoans is discussed.

Keywords: ATPases associated with diverse cellular activities; ClpB; disaggregase; heat shock protein; heat shock protein 78 (HSP78); molecular chaperones; protein folding and misfolding.

Figure 1

AAA+ superfamily features. (A) Proteins belonging to the AAA+ superfamily are characterized by the presence of an ATPase module which is 200–250 residues long with two motifs known as Walker A and Walker B. Additional motifs present are sensor 1 and sensor 2. A highly conserved motif, the second region of homology (SRH), defines the AAA family, a subset of the AAA+ family. (B) Structure from EcClpB (PDB number 4CIU; Carroni et al., 2014) from X-ray diffraction, which has a 3.5 Å resolution. Model for the 4CIU structure, in which all residues that are identical between EcClpB and Hsp78, according with the alignment shown in Figure S1, are colored in black. (C) Silhouette shape of hexameric ClpB from Cryo-EM. Shapes were drawn using Cryo-EM structures from Wendler and Saibil (2010). ClpB forms a ring-shaped hexameric structure in which the NBD1s from each monomer are on the top and the NBD2s are on the bottom. The structures are in the absence (APO; gray) and in the presence of ADP, ATP, or non-hydrolyzable AMPPNP (all in black). Superimposition with the apo form (right) indicates that the main conformational change upon either ATP or non-hydrolyzable AMPPNP binding occurs in the NBD1.

Figure 2

A human ClpB homolog (Q9H078)? (A) This gene has a signal peptide at its N-terminus for translocation (MTS), four ankyrin domains (two antiparallel α-helices followed by a β-hairpin) (in orange) and a single nucleotide binding domain (in red) containing canonical Walker A, Walker B, Sensor 1, and sensor 2 motifs. There is a rhomboid protease (PARL) cleavage site between the MTS and ankyrin like-repeats. (B) Amino acid sequence alignment of ClpB from Escherichia coli, Hsp104 from Saccharomyces cerevisiae and Q9H078 from human. Alignment created using Clustal (Sievers et al., 2011). (C) Q9H078 has GFP fluorescence precisely where mitochondria are detected, whereas the same fusion lacking the localization signal does not replicate this pattern. Left, western blot performed against human Q9H078 (dilution of 1:1000, abcam, ab87253). A homemade ECL solution was prepared for the chemiluminescent reaction, and the resulting signal was detected using a digital Chemiluminescent imaging system (GE). Right, confocal analysis of localization of the Q9H078 full length-GFP and Δ114-GFP in yeast cells. Cells were grown at 30°C and treated with the fluorescent dye MitoTracker Red (which targets active mitochondria in yeast) and subsequently fixed with 4% formaldehyde for 30 min. Cells were then washed twice with PBS (Phosphate-buffered saline) and resuspended in the same buffer and viewed by confocal microscopy (Leica, TCS SP5).