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Biomedical subjects

F U Hartl

Publications and source records attributed to F U Hartl.

At least 109 records · Page 6Linked to original sources
PubMed1991

A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1.

There is evidence to suggest that components of archaebacteria are evolutionarily related to cognates in the eukaryotic cytosol. We postulated that the major heat-shock protein of the thermophilic archaebacterium, Sulfolobus shibatae, is a molecular chaperone and that it is related to an as-yet unidentified chaperone component in the eukaryotic cytosol. Acquired thermotolerance in S. shibatae correlates with the predominant synthesis of this already abundant protein, referred to as thermophilic factor 55 (TF55). TF55 is a homo-oligomeric complex of two stacked 9-membered rings, closely resembling the 7-membered-ring complexes of the chaperonins, groEL, hsp60 and Rubisco-binding protein. The TF55 complex binds unfolded polypeptides in vitro and has ATPase activity-features consistent with its being a molecular chaperone. The primary structure of TF55, however, is not significantly related to the chaperonins. On the other hand, it is highly homologous (36-40% identity) to a ubiquitous eukaryotic protein, t-complex polypeptide-1 (TCP1). In Saccharomyces cerevisiae, TCP1 is an essential protein that may play a part in mitotic spindle formation. We suggest that TF55 in archaebacteria and TCP1 in the eukaryotic cytosol are members of a new class of molecular chaperones.

Adenosine Triphosphatases↗
PubMed1991

Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate.

Folding of two monomeric enzymes mediated by groE has been reconstituted in vitro. The groEL protein stabilizes the polypeptides in a conformation resembling the 'molten globule' state. Mg-ATP and groES then promote the acquisition of ordered tertiary structure at the surface of groEL. Folding requires the hydrolysis of about 100 ATP molecules per protein monomer. This active process of surface-mediated chain folding might represent a general mechanism for the formation of protein structure in vivo.

Adenosine Triphosphate↗
PubMed1991

Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase.

Preprotein translocation in E. coli requires ATP, the membrane electrochemical potential delta mu H+, and translocase, an enzyme with an ATPase domain (SecA) and the membrane-embedded SecY/E. Studies of translocase and proOmpA binds to the SecA domain. Second, SecA binds ATP. Third, ATP-binding energy permits translocation of approximately 20 residues of proOmpA. Fourth, ATP hydrolysis releases proOmpA. ProOmpA may then rebind to SecA and reenter this cycle, allowing progress through a series of transmembrane intermediates. In the absence of delta mu H+ or association with SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA or a membrane electrochemical potential is supplied. However, in the presence of delta mu H+ (fifth step), proOmpA rapidly completes translocation. delta mu H(+)-driven translocation is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that delta mu H+ drives translocation when ATP and proOmpA are not bound to SecA.

Adenosine Triphosphatases↗
PubMed1991

Heat shock proteins in protein folding and membrane translocation.

Constitutively expressed heat-shock proteins of the hsp60 and hsp70 families, classified as 'molecular chaperones', have important functions in the folding and intracellular sorting of newly-synthesized proteins. Recent studies of protein translocation across subcellular membranes have shown: (1) Proteins traverse membranes in extended conformations. (2) Cytosolic hsp70 proteins maintain newly-synthesized precursors destined for translocation in a loosely-folded, translocation-competent state. (3) Hsp 70 proteins on the trans-side of the membrane are required for efficient translocation. (4) In the case of mitochondria, the newly-imported polypeptides are transferred to the 'foldase' hsp60, the homologue of the E. coli groEL, which mediates their folding and oligomeric assembly. Hsp70 and hsp60 fulfill these various functions by their abilities, (1) to recognize an unknown structural element(s) which is transiently exposed in unfolded or incompletely folded polypeptides, and (2) to allow cycles of binding and (step-wise) release of the substrate protein catalyzed by ATP-hydrolysis.

Animals↗
PubMed1990

The mitochondrial chaperonin hsp60 is required for its own assembly.

Heatshock protein 60 (hsp60) in the matrix of mitochondria is essential for the folding and assembly of newly imported proteins. Hsp60 belongs to a class of structurally related chaperonins found in organelles of endosymbiotic origin and in the bacterial cytosol. Hsp60 monomers form a complex arranged as two stacked 7-mer rings. This 14-mer complex binds unfolded proteins at its surface, then seems to catalyse their folding in an ATP-dependent process. The question arises as to how such an assembly machinery is itself folded and assembled. Hsp60 subunits are encoded by a nuclear gene and translated in the cytosol as precursors which are translocated into mitochondria and proteolytically processed. In both intact cells and isolated mitochondria of the hsp60-defective yeast mutant mif4, self-assembly of newly imported wild-type subunits is not observed. Functional pre-existing hsp60 complex is required in order to form new, assembled, 14-mer. Subunits imported in vitro are assembled with a surprisingly fast half-time of 5-10 min, indicative of a catalysed reaction. These findings are further evidence that self-assembly may not be the principal mechanism by which proteins attain their functional conformation in the intact cell.

Biological Transport↗
PubMed1990

Polypeptides traverse the mitochondrial envelope in an extended state.

Most mitochondrial proteins are synthesized as precursors in the cytosol and imported through contact sites between outer and inner mitochondrial membranes. The molecular mechanism of membrane translocation of precursor proteins is largely unclear. For this report, various hybrid proteins between portions of the precursor of cytochrome b2 and the entire dihydrofolate reductase (DHFR) were accumulated in mitochondrial contact sites. We unexpectedly found that about 50 amino acid residues of the polypeptide chain in transit were sufficient to span both membranes. This suggests a linear translocation of the polypeptide chain and presents evidence for a high degree of unfolding of polypeptides traversing the mitochondrial membranes.

Biological Transport↗
PubMed1990

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane.

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.

Bacterial Outer Membrane Proteins↗
PubMed1990

Energy requirements for unfolding and membrane translocation of precursor proteins during import into mitochondria.

ATP is involved in conferring transport competence to numerous mitochondrial precursor proteins in the cytosol. Unfolded precursor proteins were found not to require ATP for import into mitochondria, suggesting a role of ATP in the unfolding of precursors. Here we report the unexpected finding that a hybrid protein containing the tightly folded passenger protein dihydrofolate reductase becomes unfolded and specifically translocated across the mitochondrial membranes independently of added ATP. Moreover, interaction of the precursor with the mitochondrial receptor components does not require ATP. The results suggest that ATP is not involved in the actual process of unfolding during membrane translocation of precursors. ATP rather appears to be necessary for preventing the formation of improper structures of precursors in the cytosol and for folding of imported polypeptides on (and release from) chaperone-like molecules in the mitochondrial matrix.

Adenosine Triphosphate↗
PubMed1990

Sorting pathways of mitochondrial inner membrane proteins.

Two distinct pathways of sorting and assembly of nuclear-encoded mitochondrial inner membrane proteins are described. In the first pathway, precursor proteins that carry amino-terminal targeting signals are initially translocated via contact sites between both mitochondrial membranes into the mitochondrial matrix. They become proteolytically processed, interact with the 60-kDa heat-shock protein hsp60 in the matrix and are retranslocated to the inner membrane. The sorting of subunit 9 of Neurospora crassa F0-ATPase has been studied as an example. F0 subunit 9 belongs to that class of nuclear-encoded mitochondrial proteins which are evolutionarily derived from a prokaryotic ancestor according to the endosymbiont hypothesis. We suggest that after import into mitochondria, these proteins follow the ancestral sorting and assembly pathways established in prokaryotes (conservative sorting). On the other hand, ADP/ATP carrier was found not to require interaction with hsp60 for import and assembly. This agrees with previous findings that the ADP/ATP carrier possesses non-amino-terminal targeting signals and uses a different import receptor to other mitochondrial precursor proteins. It is proposed that the ADP/ATP carrier represents a class of mitochondrial inner membrane proteins which do not have a prokaryotic equivalent and thus appear to follow a non-conservative sorting pathway.

Base Sequence↗
PubMed1990

Protein sorting to mitochondria: evolutionary conservations of folding and assembly.

According to the endosymbiont hypothesis, mitochondria have lost the autonomy of their prokaryotic ancestors. They have to import most of their proteins from the cytosol because the mitochondrial genome codes for only a small percentage of the polypeptides that reside in the organelle. Recent findings show that the sorting of proteins into the mitochondrial subcompartments and their folding and assembly follow principles already developed in prokaryotes. The components involved may have structural and functional equivalents in bacteria.

Amino Acid Sequence↗
PubMed1990

Protein-catalysed protein folding.

A number of proteins, termed chaperonins, have been identified as part of the mechanism of folding other proteins into their biologically active forms. The role of chaperonins appears to be twofold--to prevent illegitimate interactions with other proteins and to facilitate folding, possibly through an energy-dependent, catalytic function. Controlled overexpression of chaperonins may be of therapeutic value in manipulating human immune response and rescuing certain inherited human mutations.

Chaperonin 60↗
PubMed1989

Mitochondrial protein import.

Most mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes and are subsequently imported into mitochondria. Many precursors carry amino-terminal presequences which contain information for their targeting to mitochondria. In several cases, targeting and sorting information is also contained in non-amino-terminal portions of the precursor protein. Nucleoside triphosphates are required to keep precursors in an import-competent (unfolded) conformation. The precursors bind to specific receptor proteins on the mitochondrial surface and interact with a general insertion protein (GIP) in the outer membrane. The initial interaction of the precursor with the inner membrane requires the mitochondrial membrane potential (delta psi) and occurs at contact sites between outer and inner membranes. Completion of translocation into the inner membrane or matrix is independent of delta psi. The presequences are cleaved off by the processing peptidase in the mitochondrial matrix. In several cases, a second proteolytic processing event is performed in either the matrix or in the intermembrane space. Other modifications can occur such as the addition of prosthetic groups (e.g., heme or Fe/S clusters). Some precursors of proteins of the intermembrane space or the outer surface of the inner membrane are retranslocated from the matrix space across the inner membrane to their functional destination ('conservative sorting'). Finally, many proteins are assembled in multi-subunit complexes. Exceptions to this general import pathway are known. Precursors of outer membrane proteins are transported directly into the outer membrane in a receptor-dependent manner. The precursor of cytochrome c is directly translocated across the outer membrane and thereby reaches the intermembrane space. In addition to the general sequence of events which occurs during mitochondrial protein import, current research focuses on the molecules themselves that are involved in these processes.

Amino Acid Sequence↗