JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST001546/files/SupplemetaryData1.xlsxhttps://storage.jpostdb.org/JPST001546/files/InVitroP2020-079_20200403_QE1_HTCPAL_sand_AcOH_PE_SUSValve_2020-079_all_MMR1_2Slim.xlsxhttps://storage.jpostdb.org/JPST001546/files/20200403_QE1_HTCPAL_sand_AcOH_PE_SUSValve_2020-079_e3-A.rawhttps://storage.jpostdb.org/JPST001546/files/20200403_QE1_HTCPAL_sand_AcOH_PE_SUSValve_2020-079_e1-A.rawhttps://storage.jpostdb.org/JPST001546/files/20200403_QE1_HTCPAL_sand_AcOH_PE_SUSValve_2020-079_e2.rawhttps://storage.jpostdb.org/JPST001546/files/20180611_QE2_HTSPAL_sand_AcOH_PE_2018-150_e6.rawprimaryOK200ProteomicsTakumi KamuraSaccharomyces Cerevisiae (baker's Yeast)https://repository.jpostdb.org/entry/JPST001546Nagoya UniversityjPOSTIn yeast, mitochondria are passed on to daughter cells via the actin cable, motor protein Myo2, and adaptor protein Mmr1. They are released from the actin-myosin machinery after reaching the daughter cells. We report that Mmr1 is rapidly degraded by the ubiquitin-proteasome system in Saccharomyces cerevisiae. Redundant ubiquitin ligases Dma1 and Dma2 are responsible for Mmr1 ubiquitination. Dma1/2-mediated Mmr1 ubiquitination requires phosphorylation, most likely at S414 residue by Ste20 and Cla4. These kinases are mostly localized to the growing bud and nearly absent from mother cells, ensuring phosphorylation and ubiquitination of Mmr1 after the mitochondria enter the growing bud. In dma1Δ dma2Δ cells, transported mitochondria are first stacked at the bud-tip and then pulled back to the bud-neck. Stacked mitochondria in dma1Δ dma2Δ cells exhibit abnormal morphology, elevated respiratory activity, and increased level of reactive oxygen species, along with hypersensitivity to oxidative stresses. Collectively, spatiotemporally-regulated Mmr1 turnover guarantees mitochondrial homeostasis.Proteolysis of adaptor protein Mmr1 during budding is necessary for mitochondrial homeostasis in Saccharomyces cerevisiae.Obara Keisuke K, Yoshikawa Taku T, Yamaguchi Ryu R, Kuwata Keiko K, Nakatsukasa Kunio K, Nishimura Kohei K, Kamura Takumi TfalseProteins detected in HA-Mmr1 IP fraction; In vitro Mmr1 phosphorylationA capillary reverse-phase HPLC-MS/MS system was composed of a Dionex U3000 gradient pump equipped with a VICI Cheminert valve and Q Exactive equipped with a nano-electrospray ionization (NSI) source (AMR, Tokyo, Japan). The desalted peptides were loaded into a separation capillary C18 reverse-phase column (NTCC-360/100-3-125, 125×0.1 mm, Nikkyo Technos, Tokyo, Japan). Xcalibur 3.0.63 system (Thermo) was used to record peptide spectra over the mass range of m/z 350–1800. Repeatedly, MS spectra were recorded, followed by ten data-dependent high energy collisional dissociation (HCD) MS/MS spectra generated from the ten highest intensity precursor ions. MS/MS spectra were interpreted, and peak lists were generated using Proteome Discoverer 2.2.0.388 (Thermo). Searches were performed using the SEQUEST (Thermo) program against Saccharomyces cerevisiae (SwissProt TaxID = 559292) peptide sequence.Tue Mar 29 00:00:00 BST 2022PXD032866493235422486