Unveiling the pathway for the nuclear translocation of the large mitochondrial enzyme Pyruvate Dehydrogenase Complex (PDC)

  • Author / Creator
    Zervopoulos, Sotirios
  • Pyruvate dehydrogenase complex (PDC) is a mitochondrial enzyme that links glycolysis to oxidative phosphorylation by decarboxylating glucose-derived pyruvate to produce acetyl-CoA that is used in the Krebs cycle. PDC was initially thought to reside only in the mitochondrial matrix but we recently showed that proliferative stimuli induce its nuclear translocation, where it facilitates cell proliferation by providing acetyl-CoA towards histone acetylation. The mechanism that allows PDC to navigate throughout the cytoplasm and finally enter the nucleus is currently unknown. Protein import into the nucleus occurs through the nuclear pores (NPs) that allow the free diffusion of proteins and small metabolites of molecular weights up to 40 kDa and diameters of ~5 nm. Larger proteins require a nuclear localization sequence (NLS) that is recognized by cargo carriers before crossing the NP. PDC does not have a known NLS and its multimer size and diameter varies from small complexes that could pass through the NP to very large complexes that would not be able to cross the NP. We propose that while smaller PDC can travel (with the help of chaperones) on microtubules and reach the nucleus, entering through the NPs, larger complexes follow a novel (non-canonical) pathway that does not include NPs. Rather, it involves close contact of the mitochondria to the nucleus, allowing entrance, particularly during mitosis, in a manner similar to what is known for large viruses.
    Intracellular protein trafficking is carried out by protein carriers, including molecular chaperones, that move along the microtubules and it is facilitated by acetylation of α-tubulin. Organelles like mitochondria also move along acetylated microtubules, and tubulin acetylation may affect their trafficking. Here we show that stimuli inducing the nuclear translocation of PDC increase its colocalization with Hsp70 and acetyl-tubulin. Increased levels of tubulin acetylation facilitate the nuclear translocation of PDC. PDC can directly associate with acetylated microtubules through its E2 subunit in a manner irrespective of microtubule stability. We also found that loss of PDC but not of the main acetyl-CoA producer in the cytoplasm, ATP citrate lyase (ACLY), decreases tubulin acetylation suggesting that extramitochondrial PDC might also act as a novel tubulin acetyltransferase.
    Mitochondria are highly dynamic organelles that constantly span the cytoplasm and tether to the endoplasmic reticulum (ER) via contact points rich in mitofusin 2 (MFN2). The nuclear envelope (NE) is a continuation of the ER sharing many similarities on their protein profile. Here we show that proliferative stimuli induced a perinuclear clustering of mitochondria before we were able to detect any nuclear PDC. Perinuclear mitochondria under a transmission electron microscope formed electron-dense structures with the NE similar to those between mitochondria and the ER. We detected MFN2 on both the NE and the nucleoplasm, suggesting that it can facilitate mitochondrial tethering to the NE in a manner similar to their tethering on the ER. We also show that loss of MFN2 decreased both nuclear PDC levels and mitochondrial tethering onto the NE.
    We found that PDC is released from perinuclear mitochondria and interacts with lamin A (LMNA), which is part of the inner nuclear membrane. Loss of LMNA decreased the levels of nuclear PDC, but had no effect on SIRT6, a protein known to reside into the nucleoplasm. We found that extramitochondrial PDC embedded into the LMNA layer was not associated with the NPs. Inhibition of the trafficking through the NPs by wheat germ agglutinin (WGA) had no effect on the nuclear PDC levels after exposure to proliferative stimuli, but decreased STAT3, a protein known to enter the nucleus via the NPs. The interaction between PDC and LMNA persisted during mitosis, where LMNA is depolymerized and dispersed throughout the cytoplasm before the formation of the daughter nuclei.
    We speculate that the tethering of perinuclear mitochondria on the NE and the interaction of PDC with LMNA provides a novel mechanism for PDC to enter the nucleus in a manner that does not involve the NPs. We believe that this is the predominant mechanism for nuclear entry of PDC under proliferative conditions, without excluding some canonical PDC entrance via the NPs of smaller complexes. Our work enhances the concept of mitochondria to nucleus communication and maybe applicable to the entrance of other large proteins or viruses into the nucleus.

  • Subjects / Keywords
  • Graduation date
    Spring 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.