Mammalian and Saccharomyces cerevisiae ER-mitochondria contact site regulation by small Rab GTPases and ER folding assistants

• Author / Creator

  Herrera, Maria S

• Membrane-bound organelles allow eukaryotes to compartmentalize components and processes in a highly organized manner. Organelles can communicate with one another through
  membrane contact sites (MCS): membrane appositions 10-50nm apart. MCS were not widely accepted as bona fide sites of organelle communication until biochemical fractions of endoplasmic reticulum (ER) and mitochondria contacts were isolated and characterized. This biochemical fraction is now known as the mitochondria-associated membrane (MAM). We now know most organelles establish MCS. One of the most prominent MCS in mammalian cells is the MAM, which can occupy up to 20% of all mitochondrial surface in any one cell.

  Mitochondria-ER contact sites (MERCs) are established by protein complexes that form physical links between the ER and mitochondrial membranes. Numerous essential cellular processes occur at the MAM, including apoptosis, Ca2+ flux, mitochondrial dynamics, and autophagy. Ca2+ release from the ER plays a key role in several processes at the MAM. For example, Ca2+ is required for several Krebs cycle enzymes, so Ca2+ flux regulates respiration. Mitochondrial Ca2+ overload instead triggers apoptosis. Therefore, a fine balance must be maintained between the pro-survival and pro-death functions of the MAM. The MAM is also highly dynamic, as it can assemble and disassemble to adapt to stress or energetic demands. These observations highlight the complexity and dynamic nature of this MCS.

  In this work, we attempt to develop a better understanding of how MAM structure, tethering, and function, is regulated, and whether key mechanisms are conserved from mammals to the model organism Saccharomyces cerevisiae. We first focused on the MAM-enriched small guanosine triphosphatase (GTPase) Rab32, which regulates several aspects of the MAM, including ER-mitochondria Ca2+ flux and activation of the mitochondrial fission GTPase Dynamin related protein 1 (Drp1). Here, we investigated its poorly understood role in autophagy, a process whereby double membraned vacuoles capture cellular components to degrade them via fusion with the lysosome. We demonstrate activation of Rab32 promotes selective autophagy of the MAM, causing a decrease in several MAM-localized proteins. This process also caused a significant decrease in MERCs as assayed by electron microscopy. Additionally, we identified a new Rab32 effector: the autophagy receptor long isoform of Reticulon-3 (RTN3L). Lastly, we report dominant-active Rab32 delayed apoptosis in breast cancer cells. Given almost 25% of breast cancers have high protein levels of Rab32, we investigated if Rab32 could affect breast cancer patient outcomes. We found patients with high levels of Rab32 mRNA had a significantly worse disease outcome, as did those with high RTN3L. However, patients with high mRNA for both Rab32 and RTN3L had even worse outcomes, suggesting these proteins act synergistically in this disease.

  We also sought to investigate if mammalian mechanisms of MAM regulation are conserved in S. cerevisiae, a model organism that has been essential in furthering our understanding of MAMs. We first sought to identify a homolog for Rab32. Through phylogenetics, we identified Ypt7 as the closest homolog for Rab32 and show inactive Ypt7 increases the number of MERCs per cell. We also observed defects linked to respiration in Ypt7 mutants, suggesting Ypt7 also regulates MAM function.

  Lastly, we investigated folding assistants in S. cerevisiae given these proteins can extensively regulate MAMs in mammals. We focused on Cne1 and Eps1, the yeast homologs of the chaperone Calnexin and the reductase thioredoxin-related transmembrane protein 1 (TMX1), which regulate the activity of the sarco-endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump in mammals. Specifically, Calnexin is required to maintain SERCA2b activity while TMX1 inhibits it. Loss of these proteins therefore results in changes in Ca2+ flux, MAM tethering, and respiration. Here, we demonstrate Cne1 acts as a MAM regulator in S. cerevisiae since its loss significantly increases respiration and the number of MERCs per cells while Eps1 loss had no effect.

  In brief, this work describes a novel type of autophagy that selectively degrades the MAM, which we have proposed to call “MAMphagy”. We also demonstrate for the first time that Ypt7 and the chaperone Cne1 can regulate MAMs in S. cerevisiae. Together, these results help us better understand how MAM structure and function are regulated.

• Subjects / Keywords

  • Cell biology
  • Autophagy
  • Mitochondria-associated membrane
  • Saccharomyces cerevisiae
  • MAM

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-4ajq-0y08

• 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.