Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation.

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

GTP regulates the microtubule nucleation activity of γ-tubulin.

Both subunits of αß-tubulin that comprise the core components of microtubules bind GTP. GTP binding to α-tubulin has a structural role, whereas ß-tubulin binds and hydrolyses GTP to regulate microtubule dynamics. γ-tubulin, another member of the tubulin superfamily that seeds microtubule nucleation at microtubule-organizing centres, also binds GTP; however, the importance of this association remains elusive. To address the role of GTP binding to γ-tubulin, we systematically mutagenized the GTP contact residues in the yeast γ-tubulin Tub4. Tub4(GTP)-mutant proteins that exhibited greatly reduced GTP affinity still assembled into the small γ-tubulin complex. However, tub4(GTP) mutants were no longer viable, and had defects in interaction between γ-tubulin and αß-tubulin, decreased microtubule nucleation and defects in microtubule organization. In vitro and in vivo data show that only γ-tubulin loaded with GTP nucleates microtubules. Our results suggest that GTP recruitment to γ-tubulin enhances its interaction with αß-tubulin similarly to GTP recruitment to ß-tubulin.

A tightly regulated molecular toggle controls AAA+ disaggregase.

The ring-forming AAA+ protein ClpB cooperates with the DnaK chaperone system to refold aggregated proteins in Escherichia coli. The M domain, a ClpB-specific coiled-coil structure with two wings, motif 1 and motif 2, is essential to disaggregation, but the positioning and mechanistic role of M domains in ClpB hexamers remain unresolved. We show that M domains nestle at the ClpB ring surface, with both M-domain motifs contacting the first ATPase domain (AAA-1). Both wings contribute to maintaining a repressed ClpB activity state. Motif 2 docks intramolecularly to AAA-1 to regulate ClpB unfolding power, and motif 1 contacts a neighboring AAA-1 domain. Mutations that stabilize motif 2 docking repress ClpB, whereas destabilization leads to derepressed ClpB activity with greater unfolding power that is toxic in vivo. Our results underline the vital nature of tight ClpB activity control and elucidate a regulated M-domain toggle control mechanism.