Publications by authors named "Kanae Shirahama-Noda"

9 Publications

  • Page 1 of 1

Isoflurane induces Art2-Rsp5-dependent endocytosis of Bap2 in yeast.

FEBS Open Bio 2021 Sep 18. Epub 2021 Sep 18.

Center of Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Japan.

Although general anesthesia is indispensable during modern surgical procedures, the mechanism by which inhalation anesthetics act on the synaptic membrane at the molecular and cellular level is largely unknown. In this study, we used yeast cells to examine the effect of isoflurane, an inhalation anesthetic, on membrane proteins. Bap2, an amino acid transporter localized on the plasma membrane, was endocytosed when yeast cells were treated with isoflurane. Depletion of RSP5, an E3 ligase, prevented this endocytosis and Bap2 was ubiquitinated in response to isoflurane, indicating an ubiquitin-dependent process. Screening all the Rsp5 binding adaptors showed that Art2 plays a central role in this process. These results suggest that isoflurane affects Bap2 via an Art2-Rsp5-dependent ubiquitination system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/2211-5463.13302DOI Listing
September 2021

The PtdIns3-phosphatase MTMR3 interacts with mTORC1 and suppresses its activity.

FEBS Lett 2016 Jan 31;590(1):161-73. Epub 2015 Dec 31.

Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Japan.

Macroautophagy is a major intracellular degradation system. We previously reported that overexpression of phosphatase-deficient MTMR3, a member of the myotubularin phosphatidylinositol (PI) 3-phosphatase family, leads to induction of autophagy. In this study, we found that MTMR3 interacted with mTORC1, an evolutionarily conserved serine/threonine kinase complex, which regulates cell growth and autophagy in response to environmental stimuli. Furthermore, overexpression of MTMR3 inhibited mTORC1 activity. The N-terminal half of MTMR3, including the PH-G and phosphatase domains, was necessary and sufficient for these effects. Phosphatase-deficient MTMR3 provided more robust suppression of mTORC1 activity than wild-type MTMR3. Furthermore, phosphatase-deficient full length MTMR3 and the phosphatase domain alone were localized to the Golgi. These results suggest a new regulatory mechanism of mTORC1 in association with PI3P.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064752PMC
January 2016

Reciprocal conversion of Gtr1 and Gtr2 nucleotide-binding states by Npr2-Npr3 inactivates TORC1 and induces autophagy.

Autophagy 2014 Sep 30;10(9):1565-78. Epub 2014 Jun 30.

Center for Frontier Oral Science; Graduate School of Dentistry; Osaka University, Osaka, Japan; Graduate School of Frontier Bioscience; Osaka University; Osaka, Japan.

Autophagy is an intracellular degradation process that delivers cytosolic material to lysosomes and vacuoles. To investigate the mechanisms that regulate autophagy, we performed a genome-wide screen using a yeast deletion-mutant collection, and found that Npr2 and Npr3 mutants were defective in autophagy. Their mammalian homologs, NPRL2 and NPRL3, were also involved in regulation of autophagy. Npr2-Npr3 function upstream of Gtr1-Gtr2, homologs of the mammalian RRAG GTPase complex, which is crucial for TORC1 regulation. Both npr2∆ mutants and a GTP-bound Gtr1 mutant suppressed autophagy and increased Tor1 vacuole localization. Furthermore, Gtr2 binds to the TORC1 subunit Kog1. A GDP-bound Gtr1 mutant induced autophagy even under nutrient-rich conditions, and this effect was dependent on the direct binding of Gtr2 to Kog1. These results revealed that 2 molecular mechanisms, Npr2-Npr3-dependent GTP hydrolysis of Gtr1 and direct binding of Gtr2 to Kog1, are involved in TORC1 inactivation and autophagic induction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/auto.29397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4206535PMC
September 2014

TRAPPIII is responsible for vesicular transport from early endosomes to Golgi, facilitating Atg9 cycling in autophagy.

J Cell Sci 2013 Nov 28;126(Pt 21):4963-73. Epub 2013 Aug 28.

Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka University, 565-0871 Osaka, Japan.

Autophagy is a bulk protein-degradation process that is regulated by many factors. In this study, we quantitatively assessed the contribution of each essential yeast gene to autophagy. Of the contributing factors that we identified, we focused on the TRAPPIII complex, which was recently shown to act as a guanine-nucleotide exchange factor for the Rab small GTPase Ypt1. Autophagy is defective in the TRAPPIII mutant under nutrient-rich conditions (Cvt pathway), but starvation-induced autophagy is only partially affected. Here, we show that TRAPPIII functions at the Golgi complex to receive general retrograde vesicle traffic from early endosomes. Cargo proteins in this TRAPPIII-dependent pathway include Atg9, a transmembrane protein that is essential for autophagy, and Snc1, a SNARE unrelated to autophagy. When cells were starved, further disruption of vesicle movement from late endosomes to the Golgi caused defects in Atg9 trafficking and autophagy. Thus, TRAPPIII-dependent sorting pathways provide Atg9 reservoirs for pre-autophagosomal structure and phagophore assembly sites under nutrient-rich conditions, whereas the late endosome-to-Golgi pathway is added to these reservoirs when nutrients are limited. This clarification of the role of TRAPPIII elucidates how general membrane traffic contributes to autophagy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.131318DOI Listing
November 2013

Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages.

Nat Cell Biol 2009 Apr 8;11(4):385-96. Epub 2009 Mar 8.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.

Beclin 1, a protein essential for autophagy, binds to hVps34/Class III phosphatidylinositol-3-kinase and UVRAG. Here, we have identified two Beclin 1 associated proteins, Atg14L and Rubicon. Atg14L and UVRAG bind to Beclin 1 in a mutually exclusive manner, whereas Rubicon binds only to a subpopulation of UVRAG complexes; thus, three different Beclin 1 complexes exist. GFP-Atg14L localized to the isolation membrane and autophagosome, as well as to the ER and unknown puncta. Knockout of Atg14L in mouse ES cells caused a defect in autophagosome formation. GFP-Rubicon was localized at the endosome/lysosome. Knockdown of Rubicon caused enhancement of autophagy, especially at the maturation step, as well as enhancement of endocytic trafficking. These data suggest that the Beclin 1-hVps34 complex functions in two different steps of autophagy by altering the subunit composition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncb1846DOI Listing
April 2009

Localization of proteins and organelles using fluorescence microscopy.

Methods Mol Biol 2007 ;389:239-50

Section of Molecular Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA.

This chapter describes the different methods used for localization of proteins and organelles in Pichia pastoris. A series of plasmids and a modified immunofluorescence protocol for localization and co-localization of proteins and organelles are described. Also included are protocols for the labeling of different subcellular organelles with vital stains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-59745-456-8_17DOI Listing
December 2007

Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells.

FEBS Lett 2007 Apr 5;581(7):1417-24. Epub 2007 Mar 5.

Department of Medical Biochemistry, Shiga University of Medical Science, Seta, Otsu 520-2192, Japan.

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2007.02.064DOI Listing
April 2007

Asparagine endopeptidase is not essential for class II MHC antigen presentation but is required for processing of cathepsin L in mice.

J Immunol 2005 Jun;174(11):7066-74

Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

Class II MHC molecules survey the endocytic compartments of APCs and present antigenic peptides to CD4 T cells. In this context, lysosomal proteases are essential not only for the generation of antigenic peptides but also for proteolysis of the invariant chain to allow the maturation of class II MHC molecules. Recent studies with protease inhibitors have implicated the asparagine endopeptidase (AEP) in class II MHC-restricted Ag presentation. We now report that AEP-deficient mice show no differences in processing of the invariant chain or maturation of class II MHC products compared with wild-type mice. In the absence of AEP, presentation to primary T cells of OVA and myelin oligodendrocyte glycoprotein, two Ags that contain asparagine residues within or in proximity to the relevant epitopes was unimpaired. Cathepsin (Cat) L, a lysosomal cysteine protease essential for the development to CD4 and NK T cells, fails to be processed into its mature two-chain form in AEP-deficient cells. Despite this, the numbers of CD4 and NK T cells are normal, showing that the single-chain form of Cat L is sufficient for its function in vivo. We conclude that AEP is essential for processing of Cat L but not for class II MHC-restricted Ag presentation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.174.11.7066DOI Listing
June 2005

Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice.

J Biol Chem 2003 Aug 29;278(35):33194-9. Epub 2003 May 29.

Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.

Asparaginyl endopeptidase (AEP)/legumain, an asparagine-specific cysteine proteinase in animals, is an ortholog of plant vacuolar processing enzyme (VPE), which processes the exposed asparagine residues of various vacuolar proteins. In search for its physiological role in mammals, here we generated and characterized AEP-deficient mice. Although their body weights were significantly reduced, they were normally born and fertile. In the wild-type kidney where the expression of AEP was exceedingly high among various organs, the localization of AEP was mainly found in the lamp-2-positive late endosomes in the apical region of the proximal tubule cells. In these cells of AEP-deficient mice, the lamp-2-positive membrane structures were found to be greatly enlarged. These aberrant lysosomes, merged with the late endosomes, accumulated electron-dense and membranous materials. Furthermore, the processing of the lysosomal proteases, cathepsins B, H, and L, from the single-chain forms into the two-chain forms was completely defected in the deficient mice. Thus, the AEP deficiency caused the accumulation of macromolecules in the lysosomes, highlighting a pivotal role of AEP in the endosomal/lysosomal degradation system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M302742200DOI Listing
August 2003
-->