title: The Thermotolerant Yeast Kluyveromyces marxianus Is a Useful Organism for Structural and Biochemical Studies of Autophagy.
abstract: Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; however, because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. With the goal of expanding the scope of autophagy research, we sought to identify a novel organism with stable Atg proteins that would be advantageous for in vitro analyses. Thus, we focused on a newly isolated thermotolerant yeast strain, Kluyveromyces marxianus DMKU3-1042, to utilize as a novel system elucidating autophagy. We developed experimental methods to monitor autophagy in K. marxianus cells, identified the complete set of K. marxianus Atg homologs, and confirmed that each Atg homolog is engaged in autophagosome formation. Biochemical and bioinformatic analyses revealed that recombinant K. marxianus Atg proteins have superior thermostability and solubility as compared with S. cerevisiae Atg proteins, probably due to the shorter primary sequences of KmAtg proteins. Furthermore, bioinformatic analyses showed that more than half of K. marxianus open reading frames are relatively short in length. These features make K. marxianus proteins broadly applicable as tools for structural and biochemical studies, not only in the autophagy field but also in other fields.
authors.affiliation: From the Frontier Research Center, Tokyo Institute of Technology, Yokohama 226-8503, yamamoto-hayashi@iri.titech.ac.jp.
authors.forename: Hayashi
authors.initials: H
authors.lastname: Yamamoto
authors.affiliation: From the Frontier Research Center, Tokyo Institute of Technology, Yokohama 226-8503.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shima
authors.affiliation: the Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0812.
authors.forename: Masaya
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: the Laboratory of In Silico Functional Genomics, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501.
authors.forename: Yuh
authors.initials: Y
authors.lastname: Mochizuki
authors.affiliation: the Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube 755-8611, and.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.affiliation: From the Frontier Research Center, Tokyo Institute of Technology, Yokohama 226-8503.
authors.forename: Soichiro
authors.initials: S
authors.lastname: Kakuta
authors.affiliation: From the Frontier Research Center, Tokyo Institute of Technology, Yokohama 226-8503.
authors.forename: Chika
authors.initials: C
authors.lastname: Kondo-Kakuta
authors.affiliation: the Institute of Microbial Chemistry, Tokyo 141-0021, Japan.
authors.forename: Nobuo N
authors.initials: NN
authors.lastname: Noda
authors.affiliation: the Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0812.
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
authors.affiliation: the Laboratory of In Silico Functional Genomics, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Itoh
authors.affiliation: the Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube 755-8611, and.
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
authors.affiliation: From the Frontier Research Center, Tokyo Institute of Technology, Yokohama 226-8503, yohsumi@iri.titech.ac.jp.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ohsumi
mesh_terms: D001343:Autophagy; D019295:Computational Biology; D005470:Fluorometry; D049452:Green Fluorescent Proteins; D007716:Kluyveromyces / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016366:Open Reading Frames; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins
keywords: autophagy; bioinformatics; protein stability; recombinant protein expression; yeast