Loss of MARCH5 mitochondrial E3 ubiquitin ligase induces cellular senescence through dynamin-related protein 1 and mitofusin 1.
Mitochondria constantly divide and combine through fission and fusion activities. MARCH5, a mitochondrial E3 ubiquitin ligase, has been identified as a molecule that binds mitochondrial fission 1 protein (hFis1), dynamin-related protein 1 (Drp1) and mitofusin 2 (Mfn2), key proteins in the control of mitochondrial fission and fusion. However, how these interactions control mitochondrial dynamics, and cellular function has remained obscure. Here, we show that shRNA-mediated MARCH5 knockdown promoted the accumulation of highly interconnected and elongated mitochondria. Cells transfected with MARCH5 shRNA or a MARCH5 RING domain mutant displayed cellular enlargement and flattening accompanied by increased senescence-associated beta-galactosidase (SA-beta-Gal) activity, indicating that these cells had undergone cellular senescence. Notably, a significant increase in Mfn1 level, but not Mfn2, Drp1 or hFis1 levels, was observed in MARCH5-depleted cells, indicating that Mfn1 is a major ubiquitylation substrate. Introduction of Mfn1(T109A), a GTPase-deficient mutant form of Mfn1, into MARCH5-RNAi cells not only disrupted mitochondrial elongation, but also abolished the increase in SA-beta-Gal activity. Moreover, the aberrant mitochondrial phenotypes in MARCH5-RNAi cells were reversed by ectopic expression of Drp1, but not by hFis1, and reversion of the mitochondria morphology in MARCH5-depleted cells was accompanied by a reduction in SA-beta-Gal activity. Collectively, our data indicate that the lack of MARCH5 results in mitochondrial elongation, which promotes cellular senescence by blocking Drp1 activity and/or promoting accumulation of Mfn1 at the mitochondria.
Park
Yong-Yea
YY
Department of Biochemistry, Ajou University School of Medicine, Yeongtong-gu, Suwon 443-721, Korea.
Lee
Seungmin
S
Karbowski
Mariusz
M
Neutzner
Albert
A
Youle
Richard J
RJ
Cho
Hyeseong
H
D019943:Amino Acid Substitution; D001483:Base Sequence; D002460:Cell Line; D016922:Cellular Senescence / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008869:Microtubule-Associated Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C515578:MARCHF5 protein, human; D044767:Ubiquitin-Protein Ligases; D020558:GTP Phosphohydrolases; C470017:Mfn1 protein, human; C110768:DNM1L protein, human; D034281:Dynamins