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R Lill

Publications and source records attributed to R Lill.

At least 73 records · Page 4Linked to original sources
PubMed1995

A yeast DnaJ homologue, Scj1p, can function in the endoplasmic reticulum with BiP/Kar2p via a conserved domain that specifies interactions with Hsp70s.

Eukaryotic cells contain multiple Hsp70 proteins and DnaJ homologues. The partnership between a given Hsp70 and its interacting DnaJ could, in principle, be determined by their cellular colocalization or by specific protein-protein interactions. The yeast SCJ1 gene encodes one of several homologues of the bacterial chaperone DnaJ. We show that Scj1p is located in the lumen of the endoplasmic reticulum (ER), where it can function with Kar2p (the ER-lumenal BiP/Hsp70 of yeast). The region common to all DnaJ homologues (termed the J domain) from Scj1p can be swapped for a similar region in Sec63p, which is known to interact with Kar2p in the ER lumen, to form a functional transmembrane protein component of the secretory machinery. Thus, Kar2p can interact with two different DnaJ proteins. On the other hand, J domains from two other non-ER DnaJs, Sis1p and Mdj1p, do not function when swapped into Sec63p. However, only three amino acid changes in the Sis1p J domain render the Sec63 fusion protein fully functional in the ER lumen. These results indicate that the choice of an Hsp70 partner by a given DnaJ homologue is specified by the J domain.

Amino Acid Sequence↗
PubMed1994

A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria.

The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation.

Cytochromes↗
PubMed1994

PBP74, a new member of the mammalian 70-kDa heat shock protein family, is a mitochondrial protein.

The cloning of a cDNA encoding a new member of the highly conserved mammalian 70-kDa heat shock protein (hsp 70) family termed PBP74 was recently reported. Critical to an understanding of the function of this new hsp 70 is delineating its subcellular localization. Here we use a variety of immunological and biochemical approaches both in vitro and in vivo to demonstrate that PBP74 is imported into and resides in mitochondria. By confocal immunofluorescence microscopy PBP74 is detected in mitochondria, colocalizing with the mitochondrial 60-kDa heat shock protein. To address the inherent problem of serological cross-reactivity among the hsp70 family members, an influenza virus hemagglutinin epitope tag was introduced into the PBP74 cDNA. The epitope-tagged PBP74 protein transiently expressed in L cells localized to mitochondria. Moreover, deletion of the N-terminal 46-amino acid presequence results in a cytosolic localization of the epitope-tagged protein. Cell fractionation studies demonstrated PBP74 in purified mitochondria in a protease-protected location. After coupled transcription-translation the precursor of PBP74 is imported into isolated yeast mitochondria, where it becomes processed to the mature protein. According to a subfractionation of the mitochondria, the imported protein was found to be localized in the matrix space. Import in vitro is time- and temperature-dependent, requires matrix ATP, and is abolished upon depletion of the membrane potential across the mitochondrial inner membrane. Similarly, in mammalian cells PBP74 is synthesized as a pre-protein that requires membrane potential-dependent import into mitochondria for its maturation. Taken together, our data demonstrate that PBP74 is a mammalian mitochondrial hsp70.

Amino Acid Sequence↗
PubMed1994

Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".

We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.

Amino Acid Sequence↗
PubMed1993

Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: passage of preproteins through the intermembrane space.

The protein translocation machineries of the outer and inner mitochondrial membranes usually act in concert during translocation of matrix and inner membrane proteins. We considered whether the two machineries can function independently of each other in a sequential reaction. Fusion proteins (pF-CCHL) were constructed which contained dual targeting information, one for the intermembrane space present in cytochrome c heme lyase (CCHL) and the other for the matrix space contained in the signal sequence of the precursor of F1-ATPase beta-subunit (pF1 beta). In the absence of a membrane potential, delta psi, the fusion proteins moved into the intermembrane space using the CCHL pathway. In contrast, in the presence of delta psi they followed the pF1 beta pathway and eventually were translocated into the matrix. The fusion protein pF51-CCHL containing 51 amino acids of pF1 beta, once transported into the intermembrane space in the absence of a membrane potential, could be further chased into the matrix upon re-establishing delta psi. The sequential and independent movement of the fusion protein across the two membranes demonstrates that the translocation machineries act as distinct entities. Our results support a model in which the two translocation machineries can function independently of each other, but generally interact in a dynamic fashion to achieve simultaneous translocation across both membranes. In addition, the results provide information about the targeting sequences within CCHL. The protein does not contain a signal for retention in the intermembrane space; rather, it lacks matrix targeting information, and therefore is unable to undergo delta psi-dependent interaction with the protein translocation apparatus in the inner membrane.

Biological Transport↗
PubMed1993

Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria.

Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub-mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.

Biological Transport↗
PubMed1992

Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.

Adenosine Triphosphate↗
PubMed1990

The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.

The ATPase activity of SecA is stimulated by E. coli plasma membrane vesicles bearing SecY protein and a precursor protein such as proOmpA. This activity is termed "translocation ATPase". Liposomes alone can also stimulate SecA ATPase, but membrane proteins block this stimulation in native inner membranes. We define the stimulation of SecA ATPase by lipid as "SecA/lipid ATPase". SecA/lipid ATPase, translocation ATPase, and translocation into inner membrane vesicles require acidic phospholipids, suggesting an underlying unity of mechanism. ProOmpA and ATP stabilize liposome-bound SecA. Full SecA/lipid ATPase activity and stability are also seen when a mixture of a leader peptide and either OmpA or maltose binding protein (MBP) are added instead of proOmpA, while neither the leader peptide alone nor OmpA or MBP suffice. Cytosolic proteins in conjuction with a leader peptide are less active in this reaction, indicating that liposome-bound SecA protein recognizes both leader and mature domains.

Adenosine Triphosphatases↗
PubMed1989

Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.

Base Sequence↗
PubMed1989

SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.

We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea-treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea-treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.

Amino Acid Isomerases↗
PubMed1989

SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli.

Bacterial protein export requires two forms of energy input, ATP and the membrane electrochemical potential. Using an in vitro reaction reconstituted with purified soluble and peripheral membrane components, we can now directly measure the translocation-coupled hydrolysis of ATP. This translocation ATPase requires inner membrane vesicles, SecA protein and translocation-competent proOmpA. The stimulatory activity of membrane vesicles can be blocked by either antibody to the SecY protein or by preparing the membranes from a secY-thermosensitive strain which had been incubated at the non-permissive temperature in vivo. The SecA protein itself has more than one ATP binding site. 8-azido-ATP inactivates SecA for proOmpA translocation and for translocation ATPase, yet does not inhibit a low level of ATP hydrolysis inherent in the isolated SecA protein. These data show that the SecA protein has a central role in coupling the hydrolysis of ATP to the transfer of pre-secretory proteins across the membrane.

Adenosine Triphosphatases↗
PubMed1989

Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro.

Diverse studies of three cytoplasmic proteins of Escherichia coli--SecB, trigger factor and GroEL--have suggested that they can maintain precursor proteins in a conformation which is competent for membrane translocation. These proteins have been termed 'chaperones'. Using purified chaperone proteins and precursor protein substrates, we find that each of these chaperones can stabilize proOmpA for translocation and for the translocation-ATPase. These chaperones bind to proOmpA to form isolable complexes. SecB and GroEL will also form complexes with another exported protein, prePhoE. In contrast, these chaperones do not form stable complexes with a variety of soluble proteins such as SecA protein, bovine serum albumin, ovalbumin or ribonuclease A. While chaperones may transiently interact with soluble proteins to catalyze their folding, the stable interaction between chaperones and presecretory proteins, maintaining an open conformation which is essential for translocation, may commit these proteins to the secretion pathway.

Adenosine Triphosphatases↗
PubMed1988

Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes.

Ribosomes from Escherichia coli possess, in addition to A and P sites, a third tRNA binding site, which according to its presumed function in tRNA release during translocation has been termed the exit site. The exit site exhibits a remarkable specificity for deacylated tRNA; charged tRNA, e.g. N-AcPhe-tRNAPhe, is not bound significantly. To determine the molecular basis of this discrimination, we have measured the exit site binding affinities of a number of derivatives of tRNAPhe from E. coli, modified at the 3' end. Binding to the exit site of the tRNAPhe derivatives was measured fluorimetrically by competition with a fluorescent tRNAPhe derivative. We show here that removal of the 2' and 3' hydroxyl groups of the 3'-terminal adenosine decreases the affinity of tRNAPhe for the exit site 15 and 40-fold, respectively. Substitutions at the 3' hydroxyl group (aminoacylation, phosphorylation, cytidylation) as well as removal of the 3'-terminal adenosine (or adenylate) of tRNAPhe lower the affinity below the detection limit of 2 x 10(5) M-1, i.e. more than 100-fold. Modification of the adenine moiety (1,N6-etheno adenine) or replacement of it with other bases (cytosine, guanine) has the same dramatic effect. In contrast, the binding to both P and A sites is virtually unaffected by all of the modifications tested. These results suggest that a major fraction (at least -12 kJ/mol, probably about -17 kJ/mol) of the free energy of exit site binding of tRNAPhe (-42 kJ/mol at 20 mM-Mg2+) is contributed by the binding of the 3'-terminal adenine to the ribosome. The binding most likely entails the formation of hydrogen bonds.

Adenosine↗
PubMed1988

ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle.

We have isolated large amounts of E. coli outer-membrane protein A precursor (proOmpA). Purified proOmpA is active in membrane assembly, and this assembly is saturable with respect to the precursor protein. A proOmpA-Sepharose matrix allows affinity isolation of trigger factor, a soluble, 63,000 dalton monomeric protein that stabilizes proOmpA in assembly competent form. Comparison of trigger factor's amino-terminal sequence with those in a computer data bank and with those encoded by sec genes, as well as groEL and heat shock gene dnaK, suggests that trigger factor is encoded by a previously undescribed gene. Trigger factor and proOmpA form a 1:1 complex that can be isolated by gel filtration. Purified canine signal recognition particle (SRP) can also stabilize proOmpA for membrane insertion. This postribosomal activity of SRP suggests a unifying theme in protein translocation mechanisms.

Amino Acid Isomerases↗
PubMed1988

The "trigger factor cycle" includes ribosomes, presecretory proteins, and the plasma membrane.

Trigger factor is a soluble, 63,000 dalton protein of E. coli that stabilizes proOmpA, the precursor form of a major outer-membrane protein, in a conformation competent for in vitro membrane assembly. There is approximately one trigger factor molecule bound to each 70S ribosome isolated from cell extracts in physiological buffers. Trigger factor dissociates from ribosomes in 1.5 M LiCl and reassociates with salt-washed ribosomes in low-salt buffer. Binding is exclusively to the 50S (large) subunit, known to contain the exit domain for nascent polypeptide chains. In addition to its associations with proOmpA and ribosomes, excess trigger factor can compete with the proOmpA-trigger factor complex for a limited number of membrane sites that are essential for translocation of proOmpA. These data suggest a model of trigger factor cycling between the cytoplasm, the ribosome, presecretory proteins, and membrane receptor proteins.

Amino Acid Isomerases↗