scholarly journals Role of the chaperonin cofactor Hsp10 in protein folding and sorting in yeast mitochondria.

1994 ◽  
Vol 126 (2) ◽  
pp. 305-315 ◽  
Author(s):  
J Höhfeld ◽  
F U Hartl
Keyword(s):  
Protein Folding ◽  
Mitochondrial Protein ◽  
Temperature Sensitive ◽  
Intermembrane Space ◽  
Permissive Temperature ◽  
Loop Region ◽  
The Matrix

Protein folding in mitochondria is mediated by the chaperonin Hsp60, the homologue of E. coli GroEL. Mitochondria also contain a homologue of the cochaperonin GroES, called Hsp10, which is a functional regulator of the chaperonin. To define the in vivo role of the co-chaperonin, we have used the genetic and biochemical potential of the yeast S. cerevisiae. The HSP10 gene was cloned and sequenced and temperature-sensitive lethal hsp10 mutants were generated. Our results identify Hsp10 as an essential component of the mitochondrial protein folding apparatus, participating in various aspects of Hsp60 function. Hsp10 is required for the folding and assembly of proteins imported into the matrix compartment, and is involved in the sorting of certain proteins, such as the Rieske Fe/S protein, passing through the matrix en route to the intermembrane space. The folding of the precursor of cytosolic dihydrofolate reductase (DHFR), imported into mitochondria as a fusion protein, is apparently independent of Hsp10 function consistent with observations made for the chaperonin-mediated folding of DHFR in vitro. The temperature-sensitive mutations in Hsp10 map to a domain (residues 25-40) that corresponds to a previously identified mobile loop region of bacterial GroES and result in a reduced binding affinity of hsp10 for the chaperonin at the non-permissive temperature.

MO009NEUTRALIZATION OF UPAR WITH AN ANTI-UPAR ANTIBODY AMELIORATES RECURRENT FSGS SERA INDUCED PODOCYTE INJURY

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Flavio Vincenti ◽  
Priyanka Rashmi ◽  
Andrea Alice Da Silva ◽  
Jun Shoji ◽  
Charles Craik ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Causative Factor ◽  
Stress Fibers ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Sodium Pyruvate ◽  
Podocyte Injury

Abstract Background and Aims The role of suPAR as a biomarker and/or causative factor in the pathogenesis of recurrent FSGS remains unclear (Harel E., et al. Transplantation 2020; 104:54-60). While anti-suPAR antibodies have been shown to block suPAR induced podocyte injury in mouse models of FSGS, this beneficial effect has not yet been demonstrated in FSGS patients. We report here the inhibitor effects of 2G10, a fully human anti-suPAR antibody that blocks the interaction of suPAR with the B3 integrin on podocytes. Method The immortalized podocyte cell line was developed by transfection with the temperature sensitive SV40 T gene. These cells proliferate at the “permissive” temperature (33°C) and are considered undifferentiated. After transferring to the “nonpermissive” temperature (37°C), they enter growth arrest and by day 10-14 express markers of differentiated podocytes in vivo, such as nephrin, podocin, CD2 associated protein (CD2AP), synaptopodin, and known molecules of the slit diaphragm ZO-1, α, β, and γ-catenin, and P-cadherin. The podocytes were cultured in RPMI medium supplemented with insulin, transferrin, selenium, sodium pyruvate (ITS-A, Gibco #513000), 10% FBS and penicillin/streptomycin. After differentiation for 14 days, cells were serum starved for 1h. Serum from rFSGS patients or from a control patient was added (4% final) and cells were cultured for an additional 24h. After fixation in PFA/sucrose. actin cytoskeleton was visualized by labeling with rhodamine-conjugated phalloidin. DAPI was used for nuclei staining. Cells were imaged by confocal microscopy at 40X magnification and the number of cells with intact stress fibers were counted. For rescue of stress fibers, podocytes were cultured in the presence of a fully humanized anti uPAR antibody (2G10, 1 ug/ml; Duriseti S, J Biol Chem, 2010, 285:26878-88) or an isotype IgG control antibody (1 ug/ml). Results Sera from three patients with recurrence of FSGS after transplant were used in the study. Podocyte culture in the presence of sera from all three patients caused significant depolarization of stress fibers as determined by number of stress fiber positive cells (30%, 59% and 49% reduction with respect to untreated podocytes respectively). Treatment of podocytes with control sera did not cause any significant changes (data not shown). Culture of podocytes with patient sera in the presence of 2G10 antibody against uPAR rescued stress fibers (Fig 1A and 1B). On the other hand, a control human IgG was unable to rescue the loss of stress fibers induced by sera from recurrent FSGS patients. Conclusion The therapeutic potential of a human anti-suPAR antibody in samples from patients with recurrent FSGS has not been previously demonstrated. The in vitro findings of 2G10 antibody on preserving the stress fibers in human podocytes from the disrupting effect of the sera of patients with recurrent FSGS suggest that antibodies that block suPAR could be effective in preventing recurrence of FSGS.

scholarly journals Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria

2006 ◽  
Vol 174 (5) ◽  
pp. 631-637 ◽  
Author(s):  
Yasushi Tamura ◽  
Yoshihiro Harada ◽  
Koji Yamano ◽  
Kazuaki Watanabe ◽  
Daigo Ishikawa ◽  
...  
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Molecular Size ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Protein Import ◽  
The Matrix

Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and -16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

Dynamic organization of the mitochondrial protein import machinery

2016 ◽  
Vol 397 (11) ◽  
pp. 1097-1114 ◽  
Author(s):  
Sebastian P. Straub ◽  
Sebastian B. Stiller ◽  
Nils Wiedemann ◽  
Nikolaus Pfanner
Keyword(s):  
Mitochondrial Membrane ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Membrane Dynamics ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Precursor Proteins ◽  
Dynamic Cooperation

Abstract Mitochondria contain elaborate machineries for the import of precursor proteins from the cytosol. The translocase of the outer mitochondrial membrane (TOM) performs the initial import of precursor proteins and transfers the precursors to downstream translocases, including the presequence translocase and the carrier translocase of the inner membrane, the mitochondrial import and assembly machinery of the intermembrane space, and the sorting and assembly machinery of the outer membrane. Although the protein translocases can function as separate entities in vitro, recent studies revealed a close and dynamic cooperation of the protein import machineries to facilitate efficient transfer of precursor proteins in vivo. In addition, protein translocases were found to transiently interact with distinct machineries that function in the respiratory chain or in the maintenance of mitochondrial membrane architecture. Mitochondrial protein import is embedded in a regulatory network that ensures protein biogenesis, membrane dynamics, bioenergetic activity and quality control.

scholarly journals Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 118 (4) ◽  
pp. 609-617
Author(s):  
M Winey ◽  
M R Culbertson
Keyword(s):  
Growth Defect ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Endonuclease Activity ◽  
Temperature Dependent ◽  
Direct Role ◽  
Trna Splicing

Abstract Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.

scholarly journals Yeast 18S rRNA Dimethylase Dim1p: a Quality Control Mechanism in Ribosome Synthesis?

1998 ◽  
Vol 18 (4) ◽  
pp. 2360-2370 ◽  
Author(s):  
Denis L. J. Lafontaine ◽  
Thomas Preiss ◽  
David Tollervey
Keyword(s):  
18S Rrna ◽  
Small Subunit ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rrna Processing ◽  
Small Subunit Rrna ◽  
Enzymatic Function ◽  
Processing Steps

ABSTRACT One of the few rRNA modifications conserved between bacteria and eukaryotes is the base dimethylation present at the 3′ end of the small subunit rRNA. In the yeast Saccharomyces cerevisiae, this modification is carried out by Dim1p. We previously reported that genetic depletion of Dim1p not only blocked this modification but also strongly inhibited the pre-rRNA processing steps that lead to the synthesis of 18S rRNA. This prevented the formation of mature but unmodified 18S rRNA. The processing steps inhibited were nucleolar, and consistent with this, Dim1p was shown to localize mostly to this cellular compartment. dim1-2 was isolated from a library of conditionally lethal alleles of DIM1. In dim1-2strains, pre-rRNA processing was not affected at the permissive temperature for growth, but dimethylation was blocked, leading to strong accumulation of nondimethylated 18S rRNA. This demonstrates that the enzymatic function of Dim1p in dimethylation can be separated from its involvement in pre-rRNA processing. The growth rate ofdim1-2 strains was not affected, showing the dimethylation to be dispensable in vivo. Extracts of dim1-2 strains, however, were incompetent for translation in vitro. This suggests that dimethylation is required under the suboptimal in vitro conditions but only fine-tunes ribosomal function in vivo. Unexpectedly, when transcription of pre-rRNA was driven by a polymerase II PGKpromoter, its processing became insensitive to temperature-sensitive mutations in DIM1 or to depletion of Dim1p. This observation, which demonstrates that Dim1p is not directly required for pre-rRNA processing reactions, is consistent with the inhibition of pre-rRNA processing by an active repression system in the absence of Dim1p.

Properties of an in vitro system for studying temperature-sensitive DNA synthesis in ts A1S9 mouse L-cells

1978 ◽  
Vol 56 (6) ◽  
pp. 444-451 ◽  
Author(s):  
Jerome Humbert ◽  
Rose Sheinin
Keyword(s):  
Dna Synthesis ◽  
Buoyant Density ◽  
Velocity Sedimentation ◽  
Synthetic Activity ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Activity Restriction ◽  
L Cells

The in vitro DNA synthesis has been observed in whole cell lysates and in cytosol and nuclear fractions of wild-type (WT-4) mouse L-cells and ts A1S9 cells which exhibit temperature-sensitive (ts) DNA replication in vivo. The product, labelled with substrate 3H-labelled TTP, is resistant to alkali and has the buoyant density (1.709 g/cm3) expected for normal mouse DNA. Pulse-chase studies, in which newly made, single-stranded DNA was analyzed by velocity sedimentation in alkaline sucrose density gradients, revealed that in vitro DNA synthesis proceeds by a discontinuous mechanism. Approximately half of the DNA made in a 30-s pulse sedimented at 3–8S; the rest was very heterogeneous with S values between [Formula: see text] and 30S. After incubation for up to 300 s, a majority of the newly made DNA (>85%) sedimented as the larger, heterogeneous material, with some cosedimenting with chromosomal size DNA.The ts DNA synthesis phenotype of ts A1S9 cells is expressed in vitro. Thus, the activity of extracts of ts cells incubated at the nonpermissive (38.5 °C) temperature was commensurate with the in vivo activity. Restriction of the ts phenotype to DNA synthesis is evident in vitro since the RNA synthetic activity of lysates of temperature-inactivated ts A1S9 cells was equivalent to that of extracts obtained from cells grown at the permissive temperature (33.5 °C). The DNA synthetic activity of nuclei from WT-4 or ts A1S9 cells grown at 33.5 °C plus homologous cytosol is equivalent to that of the whole lysate. In contrast, such cytosol preparations give little, if any, enhancement of the activity of nuclei from ts A1S9 cells incubated at 38.5 °C for 16 h. The cytosol of such temperature-inactivated cells, which are almost fully effective with nuclei of control cells, produce little or no enhancement of DNA synthesis by homologous nuclei.

scholarly journals Residues of Tim44 Involved in both Association with the Translocon of the Inner Mitochondrial Membrane and Regulation of Mitochondrial Hsp70 Tethering

2008 ◽  
Vol 28 (13) ◽  
pp. 4424-4433 ◽  
Author(s):  
Dirk Schiller ◽  
Yu Chin Cheng ◽  
Qinglian Liu ◽  
William Walter ◽  
Elizabeth A. Craig
Keyword(s):  
Amino Acid ◽  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Inner Mitochondrial Membrane ◽  
The Matrix ◽  
Mitochondrial Hsp70 ◽  
Amino Acid Segment

ABSTRACT Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well established that an essential peripheral membrane protein, Tim44, tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44-mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167-amino-acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44-mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30-amino-acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J protein cochaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.

004.Control of skeletal muscle cell proliferation and differentiation

2005 ◽  
Vol 17 (9) ◽  
pp. 63
Author(s):  
M. Grounds
Keyword(s):  
Skeletal Muscle ◽  
Cell Proliferation ◽  
Multinucleated Cells ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
The Matrix ◽  
Key Issues

Skeletal muscle is formed by mononucleated precursor cells (myoblasts) that cease cell proliferation to start differentiation; this results in fusion between the myoblasts to form multinucleated cells (myotubes) that continue to differentiate (and fuse with more muscle cells) and mature into myofibres. Myogenesis has been widely used as a model to study in vitro factors controlling cell proliferation and differentiation. Condition in vitro may not reflect what happens in the more complex in vivo environment. Some of the key issues are what activates quiescent myoblasts in mature skeletal muscle in vivo, and what controls the switch between proliferation and differentiation? The role of the matrix, and molecules such as MyoD, p53, NFAT and IGF-1 will be considered.

scholarly journals Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60.

1995 ◽  
Vol 15 (5) ◽  
pp. 2654-2662 ◽  
Author(s):  
J Rassow ◽  
K Mohrs ◽  
S Koidl ◽  
I B Barthelmess ◽  
N Pfanner ◽  
...  
Keyword(s):  
Protein Folding ◽  
Cyclosporin A ◽  
Molecular Chaperones ◽  
Mitochondrial Protein ◽  
The Matrix ◽  
Inhibitory Drug ◽  
Preprotein Translocation ◽  
Cellular Compartments ◽  
Precursor Molecules

We studied the role of mitochondrial cyclophilin 20 (CyP20), a peptidyl-prolyl cis-trans isomerase, in preprotein translocation across the mitochondrial membranes and protein folding inside the organelle. The inhibitory drug cyclosporin A did not impair membrane translocation of preproteins, but it delayed the folding of an imported protein in wild-type mitochondria. Similarly, Neurospora crassa mitochondria lacking CyP20 efficiently imported preproteins into the matrix, but folding of an imported protein was significantly delayed, indicating that CyP20 is involved in protein folding in the matrix. The slow folding in the mutant mitochondria was not inhibited by cyclosporin A. Folding intermediates of precursor molecules reversibly accumulated at the molecular chaperones Hsp70 and Hsp60 in the matrix. We conclude that CyP20 is a component of the mitochondrial protein folding machinery and that it cooperates with Hsp70 and Hsp60. It is speculated that peptidyl-prolyl cis-trans isomerases in other cellular compartments may similarly promote protein folding in cooperation with chaperone proteins.

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