Mitochondrial protein quality control is crucial for the maintenance of correct mitochondrial homeostasis. It is ensured by several specific mitochondrial proteases located across the various mitochondrial subcompartments. Here, we focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 (AFG3 Like Matrix AAA Peptidase Subunit 2) and YME1L (YME1 like ATPase) of mitochondrial inner membrane AAA (ATPases Associated with diverse cellular Activities) complexes in the maintenance of mitochondrial structure and respiratory chain integrity. We demonstrate that loss of AFG3L2 and YME1L, both alone and in combination, results in diminished cell proliferation, fragmentation of mitochondrial reticulum, altered cristae morphogenesis, and defective respiratory chain biogenesis. The double AFG3L2/YME1L knockdown cells showed marked upregulation of OPA1 protein forms, with the most prominent increase in short OPA1 (optic atrophy 1). Loss of either protease led to marked elevation in OMA1 (OMA1 zinc metallopeptidase) (60 kDa) and severe reduction in the SPG7 (paraplegin) subunit of the m-AAA complex. Loss of the YME1L subunit led to an increased Drp1 level in mitochondrial fractions. While loss of YME1L impaired biogenesis and function of complex I, knockdown of AFG3L2 mainly affected the assembly and function of complex IV. Our results suggest cooperative and partly redundant functions of AFG3L2 and YME1L in the maintenance of mitochondrial structure and respiratory chain biogenesis and stress the importance of correct proteostasis for mitochondrial integrity.
- MeSH
- ATPázy spojené s různými buněčnými aktivitami genetika metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- metaloendopeptidasy genetika metabolismus MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- mitochondrie metabolismus ultrastruktura MeSH
- proliferace buněk genetika fyziologie MeSH
- proteasy závislé na ATP genetika metabolismus MeSH
- transmisní elektronová mikroskopie MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
p53 is a major cellular tumor suppressor that in addition to its nuclear, transcription-dependent activity is also known to function extranuclearly. Cellular stressors such as reactive oxygen species can promote translocation of p53 into mitochondria where it acts to protect mitochondrial genome or trigger cell death via transcription-independent manner. Here we report that the mammalian homologue of yeast mitochondrial Afg1 ATPase (LACE1) promotes translocation of p53 into mitochondria. We further show that LACE1 exhibits significant pro-apoptotic activity, which is dependent on p53, and that the protein is required for normal mitochondrial respiratory function. LACE1 physically interacts with p53 and is necessary for mitomycin c-induced translocation of p53 into mitochondria. Conversely, increased expression of LACE1 partitions p53 to mitochondria, causes reduction in nuclear p53 content and induces apoptosis. Thus, LACE1 mediates mitochondrial translocation of p53 and its transcription-independent apoptosis.
- Publikační typ
- abstrakt z konference MeSH
Mitochondrial protein homeostasis is crucial for cellular function and integrity and is therefore maintained by several classes of proteins possessing chaperone and/or proteolytic activities. In the present study, we focused on characterization of LACE1 (lactation elevated 1) function in mitochondrial protein homeostasis. LACE1 is the human homologue of yeast mitochondrial Afg1 (ATPase family gene 1) ATPase, a member of the SEC18-NSF, PAS1, CDC48-VCP, TBP family. Yeast Afg1 was shown to mediate degradation of mitochondrially encoded complex IV subunits, and, on the basis of its similarity to CDC48 (p97/VCP), it was suggested to facilitate extraction of polytopic membrane proteins. We show that LACE1, which is a mitochondrial integral membrane protein, exists as part of three complexes of approximately 140, 400 and 500 kDa and is essential for maintenance of fused mitochondrial reticulum and lamellar cristae morphology. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4 (cytochrome c oxidase 4), COX5A and COX6A, and is required for normal activity of complexes III and IV of the respiratory chain. Using affinity purification of LACE1-FLAG expressed in a LACE1-knockdown background, we show that the protein interacts physically with COX4 and COX5A subunits of complex IV and with mitochondrial inner-membrane protease YME1L. Finally, we demonstrate by ectopic expression of both K142A Walker A and E214Q Walker B mutants, that an intact ATPase domain is essential for LACE1-mediated degradation of nuclear-encoded complex IV subunits. Thus the present study establishes LACE1 as a novel factor with a crucial role in mitochondrial protein homeostasis.
- MeSH
- adenosintrifosfatasy genetika metabolismus MeSH
- cyklooxygenasy genetika metabolismus MeSH
- GTP-fosfohydrolasy genetika metabolismus MeSH
- HEK293 buňky MeSH
- konformace proteinů MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- mitochondrie ultrastruktura MeSH
- mutace MeSH
- podjednotky proteinů MeSH
- regulace genové exprese enzymů fyziologie MeSH
- RNA interference MeSH
- spotřeba kyslíku MeSH
- transport elektronů fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- ATPáza, mitochondriální proteázy,
- MeSH
- adenosintrifosfatasy metabolismus MeSH
- elektronová mikroskopie využití MeSH
- lidé MeSH
- mitochondriální membrány * enzymologie MeSH
- mitochondriální proteiny * sekrece MeSH
- mitochondriální protonové ATPasy nedostatek sekrece MeSH
- mitochondrie fyziologie MeSH
- nádorový supresorový protein p53 MeSH
- oxidativní fosforylace MeSH
- Saccharomyces cerevisiae MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.
- MeSH
- apoptóza MeSH
- genový knockdown MeSH
- GTP-fosfohydrolasy metabolismus MeSH
- lidé MeSH
- metaloendopeptidasy metabolismus MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondrie metabolismus MeSH
- NADH, NADPH oxidoreduktasy metabolismus MeSH
- proliferace buněk MeSH
- proteasy závislé na ATP metabolismus MeSH
- proteasy metabolismus MeSH
- protein - isoformy metabolismus MeSH
- respirační komplex IV metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae cytologie metabolismus MeSH
- transport elektronů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
