Institut for Biomedicin

Lars Bolund

Rapid degradation of short-chain acyl-CoA dehydrogenase variants with temperature-sensitive folding defects occurs after import into mitochondria

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Rapid degradation of short-chain acyl-CoA dehydrogenase variants with temperature-sensitive folding defects occurs after import into mitochondria. / Corydon, T J; Bross, P; Jensen, T G; Corydon, M J; Lund, T B; Jensen, Uffe Birk; Kim, J J; Gregersen, N; Bolund, L.

I: Journal of Biological Chemistry, Bind 273, Nr. 21, 22.05.1998, s. 13065-71.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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@article{04d9f3d26fb34dabbbee00c795070440,
title = "Rapid degradation of short-chain acyl-CoA dehydrogenase variants with temperature-sensitive folding defects occurs after import into mitochondria",
abstract = "Most disease-causing missense mutations in short-chain acyl-CoA dehydrogenase (SCAD) and medium-chain acyl-CoA dehydrogenase are thought to compromise the mitochondrial folding and/or stability of the mutant proteins. To address this question, we studied the biogenesis of SCAD proteins in COS-7 cells transfected with cDNA corresponding to two SCAD missense mutations, R22W (identified in a patient with SCAD deficiency) or R22C (homologous to a disease-associated R28C mutation in medium-chain acyl-CoA dehydrogenase deficiency). After cultivation at 37 degreesC the steady-state amounts of SCAD antigen and activity in extracts from cells transfected with mutant SCAD cDNAs were negligible compared with those of cells transfected with SCAD wild type cDNA, documenting the deleterious effect of the two mutations. Analysis of metabolically labeled and immunoprecipitated SCAD wild type and mutant proteins showed that the two mutant proteins were synthesized as the 44-kDa precursor form, imported into mitochondria and processed to the mature 41.7-kDa form in a normal fashion. However, the intramitochondrial level of matured mutant SCAD proteins decreased rapidly to very low levels, indicating a rapid degradation of the mutant proteins at 37 degreesC. A rapid initial elimination phase was also observed following cultivation at 26 degreesC; however, significantly higher amounts of metabolically labeled and immunoprecipitated mature mutant SCAD proteins remained detectable. This corresponds well with the appreciable steady-state levels of SCAD mutant enzyme activity observed at 26 degreesC. In addition, confocal laser scanning microscopy of immunostained cells showed that the SCAD mutant proteins were localized intramitochondrially. Together, these results show that newly synthesized SCAD R22W and R22C mutant proteins are imported and processed in the mitochondrial matrix, but that a fraction of the proteins is rapidly eliminated by a temperature-dependent degradation mechanism. Thermal stability profiles of wild type and mutant enzymes revealed no difference between the two mutants and the wild type protein. Furthermore, the turnover of the SCAD mutant enzymes in intact cells was comparable to that of the wild type, indicating that the rapid degradation of the mutant SCAD proteins is not due to lability of the correctly folded tetrameric structure but rather to elimination of partly folded or misfolded proteins along the folding pathway.",
keywords = "Acyl-CoA Dehydrogenase, Acyl-CoA Dehydrogenases, Animals, Biological Transport, COS Cells, Humans, Hydrolysis, Microscopy, Confocal, Mitochondria, Mutagenesis, Protein Folding, Temperature",
author = "Corydon, {T J} and P Bross and Jensen, {T G} and Corydon, {M J} and Lund, {T B} and Jensen, {Uffe Birk} and Kim, {J J} and N Gregersen and L Bolund",
year = "1998",
month = may,
day = "22",
language = "English",
volume = "273",
pages = "13065--71",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "21",

}

RIS

TY - JOUR

T1 - Rapid degradation of short-chain acyl-CoA dehydrogenase variants with temperature-sensitive folding defects occurs after import into mitochondria

AU - Corydon, T J

AU - Bross, P

AU - Jensen, T G

AU - Corydon, M J

AU - Lund, T B

AU - Jensen, Uffe Birk

AU - Kim, J J

AU - Gregersen, N

AU - Bolund, L

PY - 1998/5/22

Y1 - 1998/5/22

N2 - Most disease-causing missense mutations in short-chain acyl-CoA dehydrogenase (SCAD) and medium-chain acyl-CoA dehydrogenase are thought to compromise the mitochondrial folding and/or stability of the mutant proteins. To address this question, we studied the biogenesis of SCAD proteins in COS-7 cells transfected with cDNA corresponding to two SCAD missense mutations, R22W (identified in a patient with SCAD deficiency) or R22C (homologous to a disease-associated R28C mutation in medium-chain acyl-CoA dehydrogenase deficiency). After cultivation at 37 degreesC the steady-state amounts of SCAD antigen and activity in extracts from cells transfected with mutant SCAD cDNAs were negligible compared with those of cells transfected with SCAD wild type cDNA, documenting the deleterious effect of the two mutations. Analysis of metabolically labeled and immunoprecipitated SCAD wild type and mutant proteins showed that the two mutant proteins were synthesized as the 44-kDa precursor form, imported into mitochondria and processed to the mature 41.7-kDa form in a normal fashion. However, the intramitochondrial level of matured mutant SCAD proteins decreased rapidly to very low levels, indicating a rapid degradation of the mutant proteins at 37 degreesC. A rapid initial elimination phase was also observed following cultivation at 26 degreesC; however, significantly higher amounts of metabolically labeled and immunoprecipitated mature mutant SCAD proteins remained detectable. This corresponds well with the appreciable steady-state levels of SCAD mutant enzyme activity observed at 26 degreesC. In addition, confocal laser scanning microscopy of immunostained cells showed that the SCAD mutant proteins were localized intramitochondrially. Together, these results show that newly synthesized SCAD R22W and R22C mutant proteins are imported and processed in the mitochondrial matrix, but that a fraction of the proteins is rapidly eliminated by a temperature-dependent degradation mechanism. Thermal stability profiles of wild type and mutant enzymes revealed no difference between the two mutants and the wild type protein. Furthermore, the turnover of the SCAD mutant enzymes in intact cells was comparable to that of the wild type, indicating that the rapid degradation of the mutant SCAD proteins is not due to lability of the correctly folded tetrameric structure but rather to elimination of partly folded or misfolded proteins along the folding pathway.

AB - Most disease-causing missense mutations in short-chain acyl-CoA dehydrogenase (SCAD) and medium-chain acyl-CoA dehydrogenase are thought to compromise the mitochondrial folding and/or stability of the mutant proteins. To address this question, we studied the biogenesis of SCAD proteins in COS-7 cells transfected with cDNA corresponding to two SCAD missense mutations, R22W (identified in a patient with SCAD deficiency) or R22C (homologous to a disease-associated R28C mutation in medium-chain acyl-CoA dehydrogenase deficiency). After cultivation at 37 degreesC the steady-state amounts of SCAD antigen and activity in extracts from cells transfected with mutant SCAD cDNAs were negligible compared with those of cells transfected with SCAD wild type cDNA, documenting the deleterious effect of the two mutations. Analysis of metabolically labeled and immunoprecipitated SCAD wild type and mutant proteins showed that the two mutant proteins were synthesized as the 44-kDa precursor form, imported into mitochondria and processed to the mature 41.7-kDa form in a normal fashion. However, the intramitochondrial level of matured mutant SCAD proteins decreased rapidly to very low levels, indicating a rapid degradation of the mutant proteins at 37 degreesC. A rapid initial elimination phase was also observed following cultivation at 26 degreesC; however, significantly higher amounts of metabolically labeled and immunoprecipitated mature mutant SCAD proteins remained detectable. This corresponds well with the appreciable steady-state levels of SCAD mutant enzyme activity observed at 26 degreesC. In addition, confocal laser scanning microscopy of immunostained cells showed that the SCAD mutant proteins were localized intramitochondrially. Together, these results show that newly synthesized SCAD R22W and R22C mutant proteins are imported and processed in the mitochondrial matrix, but that a fraction of the proteins is rapidly eliminated by a temperature-dependent degradation mechanism. Thermal stability profiles of wild type and mutant enzymes revealed no difference between the two mutants and the wild type protein. Furthermore, the turnover of the SCAD mutant enzymes in intact cells was comparable to that of the wild type, indicating that the rapid degradation of the mutant SCAD proteins is not due to lability of the correctly folded tetrameric structure but rather to elimination of partly folded or misfolded proteins along the folding pathway.

KW - Acyl-CoA Dehydrogenase

KW - Acyl-CoA Dehydrogenases

KW - Animals

KW - Biological Transport

KW - COS Cells

KW - Humans

KW - Hydrolysis

KW - Microscopy, Confocal

KW - Mitochondria

KW - Mutagenesis

KW - Protein Folding

KW - Temperature

M3 - Journal article

C2 - 9582344

VL - 273

SP - 13065

EP - 13071

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 21

ER -