Biomedical subjects

U Fünfschilling

Publications and source records attributed to U Fünfschilling.

4 recordsLinked to original sources

PubMed2001

RAC, a stable ribosome-associated complex in yeast formed by the DnaK-DnaJ homologs Ssz1p and zuotin.

The yeast cytosol contains multiple homologs of the DnaK and DnaJ chaperone family. Our current understanding of which homologs functionally interact is incomplete. Zuotin is a DnaJ homolog bound to the yeast ribosome. We have now identified the DnaK homolog Ssz1p/Pdr13p as zuotin's partner chaperone. Zuotin and Ssz1p form a ribosome-associated complex (RAC) that is bound to the ribosome via the zuotin subunit. RAC is unique among the eukaryotic DnaK-DnaJ systems, as the 1:1 complex is stable, even in the presence of ATP or ADP. In vitro, RAC stimulates the translocation of a ribosome-bound mitochondrial precursor protein into mitochondria, providing evidence for its chaperone-like effect on nascent chains. In agreement with the existence of a functional complex, deletion of each RAC subunit resulted in a similar phenotype in vivo. However, overexpression of zuotin partly rescued the growth defect of the Delta ssz1 strain, whereas overexpression of Ssz1p did not affect the Delta zuo1 strain, suggesting a pivotal function for the DnaJ homolog.

Cytosol↗

PubMed1999

Nascent polypeptide-associated complex stimulates protein import into yeast mitochondria.

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide-associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.

Biological Transport↗

PubMed1998

Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10.

The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo.

Chaperonin 10↗

PubMed1996

Functional analysis of the MAP2 repeat domain.

The neuronal microtubule-associated protein MAP2 binds to microtubules via a domain near its C terminus containing a set of 3 or 4 imperfect repeats of a 31 amino acid motif. Using naturally occurring and mutated forms of the molecule containing between 1 and 4 repeats we have examined the contribution that these repeats make to MAP2 function and explored the significance of their repetition. The experiments utilised the short 3- and 4-repeat splice variants MAP2c and MAP2d that are expressed in developing neurons and in glia respectively, and mutant 1- and 2-repeat versions that were produced by using in vitro mutagenesis to remove further 31 amino acid units while leaving the rest of the molecule unaltered. The properties of these MAP2 variants were compared both with respect to their influence on microtubules in transfected non-neuronal cells and their ability to promote microtubule assembly in vitro. We found that each of the known effects of MAP2, including the bundling of microtubules and induction of process formation in living cells, are expressed by the 1-repeat form MAP2c3, which contains only the third repeat (R3). A second 1-repeat form, MAP2c4, which contains only R4, interacts more weakly with tubulin in vitro and does not bind to microtubules in transfected cells. The microtubule-related properties of MAP2 thus arise mainly from a single predominant repeat unit, R3. In vitro assembly experiments showed that the primary effect of all the repeats is to lower the critical concentration of tubulin required for microtubule assembly but that they differ greatly in potency. The results did not reveal a separate function related to the repetition of the repeat motifs, but instead suggest that its purpose is to tailor the efficiency of MAP2 to the cellular environment in which it has to function.

Amino Acid Sequence↗