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Cited by 173Heat shock proteins researchMitochondrial Function and PathologyNeurological diseases and metabolism

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Mt-Hsp70 Homolog, Ssc2p, Required for Maturation of Yeast Frataxin and Mitochondrial Iron Homeostasis

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Knight, Simon A.B., Sepuri, Naresh Babu V., Pain, Debkumar, Dancis, Andrew·1998·Journal of Biological Chemistry
DOI 10.1074/jbc.273.29.18389
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Abstract

Here we show that the yeast mitochondrial chaperone Ssc2p, a homolog of mt-Hsp70, plays a critical role in mitochondrial iron homeostasis. Yeast with ssc2-1 mutations were identified by a screen for altered iron-dependent gene regulation and mitochondrial dysfunction. These mutants exhibit increased cellular iron uptake, and the iron accumulates exclusively within mitochondria. Yfh1p is homologous to frataxin, the human protein implicated in the neurodegenerative disease, Friedreich's ataxia. Like mutants of yfh1, ssc2-1 mutants accumulate vast quantities of iron in mitochondria. Furthermore, using import studies with isolated mitochondria, we demonstrate a specific role for Ssc2p in the maturation of Yfh1p within this organelle. This function for a mitochondrial Hsp70 chaperone is likely to be conserved, implying that a human homolog of Ssc2p may be involved in iron homeostasis and in neurodegenerative disease.

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FIG. 1 Phenotypes of the ssc2 -1 mutants. A , mtDNA inactivation in strain 341-5B ( ssc2 -1 ). Strains 81rho 0 ( 1. rho 0 ) and 341-5B ( 2. ssc2 -1 ) were crossed and zygotes were manipulated. Other haploid controls were 191-33C ( 3. ssc2 -1 ), 61 ( 4. WT ), 61rho 0 ( 5. rho 0 ). Haploids and diploid clones arising from the cross were transferred to YE ( Ethanol ) or YPD ( Glucose ). Failure of the diploid clones to grow on ethanol plates is diagnostic of mtDNA inactivation in the parental strain, 341-5B ( ssc2 -1 ). B , growth characteristics of the mutants: temperature and iron sensitivity. Diploid x191-36 was sporulated, and spore clones carrying the mutant allele ssc2 -1 ( A , B ) or the wild-type allele SSC2 ( C , D ) were examined for growth on YPD agar at different temperatures (30 °C, 23 °C, 37 °C) or for growth on SD medium containing 1 mM ferrozine ( Chelator , no added iron; Iron , 250 m M ferric ammonium added). The relative concentrations of the inocula spotted onto the plates are indicated by 1 (10 3 cells/10 m l) and 1:10. The wild-type clone C appeared pigmented because of a genetic trait unlinked to SSC2 . C , high affinity cellular iron uptake increased in the ssc1 -2 mutants. The spore clones were grown to logarithmic phase in YPD, and iron uptake was assayed using 1 m M 55 Fe radionuclide in 50 mM sodium citrate buffer, pH 6.5, as described. Data are the mean 6 S.D. of triplicate measurements. D , mitochondrial iron content increased in the ssc1 -2 mutants. Strains 61 ( WT ), 61rho 0 ( rho 0 ), and 191-33C ( ssc2 -1 ) were grown in media with 0.9, 1.8, or 5 m M iron, and the mitochondrial iron content was assayed as described.
FIG. 1 Phenotypes of the ssc2 -1 mutants. A , mtDNA inactivation in strain 341-5B ( ssc2 -1 ). Strains 81rho 0 ( 1. rho 0 ) and 341-5B ( 2. ssc2 -1 ) were crossed and zygotes were manipulated. Other haploid controls were 191-33C ( 3. ssc2 -1 ), 61 ( 4. WT ), 61rho 0 ( 5. rho 0 ). Haploids and diploid clones arising from the cross were transferred to YE ( Ethanol ) or YPD ( Glucose ). Failure of the diploid clones to grow on ethanol plates is diagnostic of mtDNA inactivation in the parental strain, 341-5B ( ssc2 -1 ). B , growth characteristics of the mutants: temperature and iron sensitivity. Diploid x191-36 was sporulated, and spore clones carrying the mutant allele ssc2 -1 ( A , B ) or the wild-type allele SSC2 ( C , D ) were examined for growth on YPD agar at different temperatures (30 °C, 23 °C, 37 °C) or for growth on SD medium containing 1 mM ferrozine ( Chelator , no added iron; Iron , 250 m M ferric ammonium added). The relative concentrations of the inocula spotted onto the plates are indicated by 1 (10 3 cells/10 m l) and 1:10. The wild-type clone C appeared pigmented because of a genetic trait unlinked to SSC2 . C , high affinity cellular iron uptake increased in the ssc1 -2 mutants. The spore clones were grown to logarithmic phase in YPD, and iron uptake was assayed using 1 m M 55 Fe radionuclide in 50 mM sodium citrate buffer, pH 6.5, as described. Data are the mean 6 S.D. of triplicate measurements. D , mitochondrial iron content increased in the ssc1 -2 mutants. Strains 61 ( WT ), 61rho 0 ( rho 0 ), and 191-33C ( ssc2 -1 ) were grown in media with 0.9, 1.8, or 5 m M iron, and the mitochondrial iron content was assayed as described.CC-BY · From the PDF
FIG. 2 Electron microscopy showing iron-laden mitochondria. A , ssc2 -1 cells from strain 191-33C with accumulated iron visible as dense bodies. B , higher magnification ( 3 11,000) view of a single ssc2 -1 cell showing accumulated iron within mitochondria. Mitochondria ( m ) and nucleus ( n ) are indicated. C , wild-type cell from strain 61. D , single ssc2 -1 cell with iron-laden mitochondria ringing the nucleus. The calibration bar in each panel represents 1 m m.
FIG. 2 Electron microscopy showing iron-laden mitochondria. A , ssc2 -1 cells from strain 191-33C with accumulated iron visible as dense bodies. B , higher magnification ( 3 11,000) view of a single ssc2 -1 cell showing accumulated iron within mitochondria. Mitochondria ( m ) and nucleus ( n ) are indicated. C , wild-type cell from strain 61. D , single ssc2 -1 cell with iron-laden mitochondria ringing the nucleus. The calibration bar in each panel represents 1 m m.CC-BY · From the PDF
FIG. 3 Yfh1 preprotein processing impaired in ssc2 -1 mitochondria. Import of urea-denatured precursors of Yfh1 ( A ), Yfh1Protein A ( B ), or Put2 ( C ) were evaluated in mitochondria purified from wild-type ( WT ) or ssc2 -1 ( M ) strains. Import reactions were allowed to proceed for 5 min (5 9 ) or 15 min (15 9 ) at 20 °C. Where indicated, unimported precursor was digested by trypsin. p , i , and m signify the precursor, the intermediate, and the mature form, respectively. Lane 1 in each panel ( Std ) indicates 35% of the precursor used per import assay.
FIG. 3 Yfh1 preprotein processing impaired in ssc2 -1 mitochondria. Import of urea-denatured precursors of Yfh1 ( A ), Yfh1Protein A ( B ), or Put2 ( C ) were evaluated in mitochondria purified from wild-type ( WT ) or ssc2 -1 ( M ) strains. Import reactions were allowed to proceed for 5 min (5 9 ) or 15 min (15 9 ) at 20 °C. Where indicated, unimported precursor was digested by trypsin. p , i , and m signify the precursor, the intermediate, and the mature form, respectively. Lane 1 in each panel ( Std ) indicates 35% of the precursor used per import assay.CC-BY · From the PDF
FIG. 4 Model for the involvement of Ssc2p in Yfh1 preprotein processing and iron homeostasis. Ovals represent yeast cells, the wild-type on the left and the mutant ( ssc2 -1 ) on the right . The box enclosed by the double line represents the mitochondria. Yellow indicates protein trafficking and processing pathways (Yfh1p translocation and maturation). Red indicates iron trafficking pathways, with boxes showing steady state iron pools. Blue indicates regulatory loops, with Aft1p controlling uptake at the plasma membrane and Yfh1p/Ssc2p controlling iron accumulation within the mitochondria.
FIG. 4 Model for the involvement of Ssc2p in Yfh1 preprotein processing and iron homeostasis. Ovals represent yeast cells, the wild-type on the left and the mutant ( ssc2 -1 ) on the right . The box enclosed by the double line represents the mitochondria. Yellow indicates protein trafficking and processing pathways (Yfh1p translocation and maturation). Red indicates iron trafficking pathways, with boxes showing steady state iron pools. Blue indicates regulatory loops, with Aft1p controlling uptake at the plasma membrane and Yfh1p/Ssc2p controlling iron accumulation within the mitochondria.CC-BY · From the PDF

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  1. Dancis, 1990, Mol. Cell. Biol., 10, 2294

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Metadata

Authors
Knight, S. A. B., Sepuri, N. B. V., Pain, D., & Dancis, A.
Affiliations
University of Pennsylvania (US)
Corresponding author
Dancis, Andrew
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
1998-07
Volume
273
Issue
29
Pages
18389-18393
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.273.29.18389
OpenAlex
W1976493565
Cited by
173
References
26
Citations by year
2024: 4 · 2023: 2 · 2022: 1 · 2021: 2 · 2020: 1 · 2019: 4 · 2018: 3 · 2017: 7 · 2016: 2 · 2015: 5 · 2014: 6 · 2013: 2 · 2012: 8
FWCI
6.17
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • American Heart Association
  • W. W. Smith Charitable Trust
  • University of Pennsylvania
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Neuroscience › Neurology › Neurological diseases and metabolism
Keywords
mitochondrial Hsp70, frataxin, mitochondrial iron homeostasis, protein maturation, mitochondrial chaperone, Friedreich's ataxia, iron accumulation, iron absorption, mitochondrial dysfunction, Saccharomyces cerevisiae
MeSH terms
Frataxin, Fungal Proteins, Homeostasis, Iron, Mitochondria, Models, Biological, Saccharomyces cerevisiae, Gene Library, Mutagenesis, Site-Directed, Phosphotransferases (Alcohol Group Acceptor), Molecular Chaperones, HSP70 Heat-Shock Proteins, Mitochondrial Proteins, Saccharomyces cerevisiae Proteins, Iron-Binding Proteins

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