We also observed that GroES, a heptameric ring of ∼10 kDa subunits, bound like a lid over one end of the GroEL cylinder, concomitant with major conformational changes in the interacting GroEL ring. ![]() The new images revealed that unfolded substrate protein binds in the ring center. As shown earlier, the ∼800 kDa GroEL complex consists of two stacked, heptameric rings. We next investigated the GroEL system by electron microscopy in collaboration with Wolfgang Baumeister ( Langer et al., 1992a). In 1991 I took on a faculty position at Sloan-Kettering Cancer Center in Manhattan in the new department of Jim Rothman. In the presence of GroES, GroEL released the substrate protein in a more folded, less aggregation-prone state. Fluorescence spectroscopy revealed that GroEL binds nonnative proteins in a loosely folded, “molten globule”–like conformation, preventing their aggregation. To be sure that we measured folding, not assembly, Jörg Martin in my lab chose monomeric proteins (DHFR and rhodanese) as substrates for reconstitution experiments ( Martin et al., 1991). These findings in 1989 established the new paradigm of chaperone-assisted protein folding (reviewed in Hartl, 1996).īut how did the chaperonins work? George Lorimer made the next advance by reconstituting bacterial Rubisco, a dimeric enzyme, from the denatured state with the help of GroEL and its cofactor GroES ( Goloubinoff et al., 1989). Hence the defects in oligomeric assembly observed in the mif4 mitochondria resulted from the failure of protein subunits to fold. We concluded that Hsp60-and by analogy the other chaperonins-mediated protein folding. Formation of folded DHFR occurred upon readdition of ATP concomitantly with release from Hsp60. Instead, the newly imported protein associated with Hsp60 in an unfolded state that was stabilized under ATP limiting conditions. Denatured DHFR will refold spontaneously in vitro, but strikingly, this was not what we observed in mitochondria. In these experiments, carried out with my student Joachim Ostermann, we targeted the monomeric protein dihydrofolate reductase (DHFR) to mitochondria ( Ostermann et al., 1989). While our initial findings on Hsp60 were consistent with such a role, a second generation of experiments soon revealed the basic function of the chaperonin in polypeptide chain folding. GroEL was known as a genetic host factor in phage propagation, and RBP had been observed to bind unassembled subunits of the enzyme Rubisco (Barraclough and Elis, 1980), suggesting a role in mediating protein assembly.įIGURE 1: Art Horwich (right) and myself in March 1991 taking a walk in my parents’ village in the northern part of the Black Forest. Intriguingly, the mif4 mutation mapped to the nuclear gene encoding mitochondrial Hsp60, the homologue of Escherichia coli GroEL and the Rubisco subunit-binding protein (RBP) of chloroplasts-large complexes called “chaperonins” ( Hemmingsen et al., 1988). We embarked on an exciting collaboration and found that mif4 mitochondria remained import-competent, but the imported proteins failed to assemble into their respective oligomeric complexes ( Cheng et al., 1989). One of his temperature-sensitive mutants, called mif4 (for mitochondrial import function 4), was particularly puzzling. I was fortunate that Walter put me in touch with Art Horwich ( Figure 1), who had conducted a genetic screen in yeast to identify cellular machinery involved in mitochondrial protein import. How then would these proteins fold and assemble inside the organelle? I found myself at the right place at the right time to address this fascinating problem. Incidentally, I am writing these lines on our 30th wedding anniversary.Īround the time of my arrival in the Munich lab it became clear that proteins had to be unfolded in order to translocate from the cytosol across the mitochondrial membranes. His mentorship turned out to be critical, not only professionally but also personally, as he allowed me to attend a molecular biology summer school where I met my future wife, Manajit. Walter was famous for his studies on how mitochondria import newly synthesized proteins from the cytosol. I was lucky that Walter Neupert at Munich University was invited as an external reviewer of my thesis, and this led to my joining his group in 1985. ![]() One of our findings was that the peroxisomal membrane system could be induced by thyroid hormones. My doctoral thesis in the biochemistry department under the guidance of Wilhelm Just focused on the functions of peroxisomes in the rat liver. I was introduced to research as a young medical student at Heidelberg University. These early findings would have a lasting impact on our scientific careers. I have fond memories of our close collaboration and of the excitement we felt when our experiments provided first evidence of protein folding as a chaperone-assisted process.
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