Katja Siegers

A novel protein complex promoting formation of functional alpha- and gamma-tubulin.

We describe the identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with tub4‐1, encoding a mutated yeast γ‐tubulin. The cytoplasmic Gim proteins encoded by these GIM genes are present in common complexes as judged by co‐immunoprecipitation and gel filtration experiments. The disruption of any of these genes results in similar phenotypes: the gim null mutants are synthetically lethal with tub4‐1 and super‐sensitive towards the microtubule‐depolymerizing drug benomyl. All except Δgim4 are cold‐sensitive and their microtubules disassemble at 14°C. The Gim proteins have one function related to α‐tubulin and another to Tub4p, supported by the finding that the benomyl super‐sensitivity is caused by a reduced level of α‐tubulin while the synthetic lethality with tub4‐1 is not. In addition, GIM1/YKE2 genetically interacts with two distinct classes of genes, one of which is involved in tubulin folding and the other in microtubule nucleation. We show that the Gim proteins are important for Tub4p function and bind to overproduced Tub4p. The mammalian homologues of GIM1/YKE2 and GIM2/PAC10 rescue the synthetically lethal phenotype with tub4‐1 as well as the cold‐sensitivity and benomyl super‐sensitivity of the yeast deletion mutants. We suggest that the Gim proteins form a protein complex that promotes formation of functional α‐ and γ‐tubulin.

Compartmentation of protein folding in vivo: sequestration of non‐native polypeptide by the chaperonin–GimC system

The functional coupling of protein synthesis and chaperone‐assisted folding in vivo has remained largely unexplored. Here we have analysed the chaperonin‐dependent folding pathway of actin in yeast. Remarkably, overexpression of a heterologous chaperonin which traps non‐native polypeptides does not interfere with protein folding in the cytosol, indicating a high‐level organization of folding reactions. Newly synthesized actin avoids the chaperonin trap and is effectively channelled from the ribosome to the endogenous chaperonin TRiC. Efficient actin folding on TRiC is critically dependent on the hetero‐oligomeric co‐chaperone GimC. By interacting with folding intermediates and with TRiC, GimC accelerates actin folding at least 5‐fold and prevents the premature release of non‐native protein from TRiC. We propose that TRiC and GimC form an integrated ‘folding compartment’ which functions in cooperation with the translation machinery. This compartment sequesters newly synthesized actin and other aggregation‐sensitive polypeptides from the crowded macromolecular environment of the cytosol, thereby allowing their efficient folding.

MtGimC, a novel archaeal chaperone related to the eukaryotic chaperonin cofactor GimC/prefoldin

Group II chaperonins in the eukaryotic and archaeal cytosol assist in protein folding independently of the GroES‐like cofactors of eubacterial group I chaperonins. Recently, the eukaryotic chaperonin was shown to cooperate with the hetero‐oligomeric protein complex GimC (prefoldin) in folding actin and tubulins. Here we report the characterization of the first archaeal homologue of GimC, from Methanobacterium thermoautotrophicum. MtGimC is a hexamer of 87 kDa, consisting of two α and four β subunits of high α‐helical content that are predicted to contain extended coiled coils and represent two evolutionarily conserved classes of Gim subunits. Reconstitution experiments with MtGimC suggest that two subunits of the α class (archaeal Gimα and eukaryotic Gim2 and 5) form a dimer onto which four subunits of the β class (archaeal Gimβ and eukaryotic Gim1, 3, 4 and 6) assemble. MtGimα and β can form hetero‐complexes with yeast Gim subunits and MtGimβ partially complements yeast strains lacking Gim1 and 4. MtGimC is a molecular chaperone capable of stabilizing a range of non‐native proteins and releasing them for subsequent chaperonin‐assisted folding. In light of the absence of Hsp70 chaperones in many archaea, GimC may fulfil an ATP‐independent, Hsp70‐like function in archaeal de novo protein folding.

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes.

The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood. Here, we show that a group of WD40 β‐propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state. TRiC cooperates in the folding of these proteins with the ribosome‐associated heat shock protein (Hsp)70 chaperones Ssb1/2p. In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co‐chaperone GimC/prefoldin for efficient transfer to the chaperonin. GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability. These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC‐assisted folding.

L25 functions as a conserved ribosomal docking site shared by nascent chain‐associated complex and signal‐recognition particle

The nascent chain-associated complex (NAC) is a dimeric protein complex of archaea and eukarya that interacts with ribosomes and translating polypeptide chains. We show that, in yeast, NAC and the signal-recognition particle (SRP) share the universally conserved ribosomal protein L25 as a docking site, which is in close proximity to the ribosomal exit tunnel. The amino-terminal segment of beta-NAC was found to be required for L25 binding. Purified NAC can prevent protein aggregation in vitro and thus shows certain properties of a molecular chaperone. Interestingly, the alpha-subunit of NAC interacts with the 54 kDa subunit of SRP. Consistent with a regulatory role of NAC in protein translocation into the endoplasmic reticulum (ER), we find that deletion of NAC results in an induction of the ER stress-response pathway. These results identify L25 as a conserved interaction platform for specific cytosolic factors that guide nascent polypeptides to their proper cellular destination.