ABSTRACT
The heteromeric complex of the two AAA+ ATPases PEX1 and PEX6 is involved in the export of the monoubiquitinated import receptor PEX5 from the peroxisomal membrane. Mutations in this complex make up for over 60% of the patients with Peroxisomal Biogenesis Disorders. To have better options for the treatment of the milder mutations we purified the human PEX1/PEX6 complex after overexpression of plasmids encoding tagged proteins from HEK293TT cells. We used a combination of a HisTrap Column (Ni-NTA chromatography) and a Strep-Tactin®XT cartridge for small-scale purification of the complex using the His-tag of PEX1 and the Strep-tagII of PEX6.
Subject(s)
ATPases Associated with Diverse Cellular Activities , Recombinant Fusion Proteins , HEK293 Cells , Humans , Chromatography, Affinity/methods , Recombinant Fusion Proteins/isolation & purification , Plasmids/genetics , ATPases Associated with Diverse Cellular Activities/isolation & purification , Transfection , Cell SeparationABSTRACT
The fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3-H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3-H4 loading by utilizing ATP.
Subject(s)
ATPases Associated with Diverse Cellular Activities/ultrastructure , Histone Chaperones/ultrastructure , Nucleosomes/metabolism , Schizosaccharomyces pombe Proteins/ultrastructure , ATPases Associated with Diverse Cellular Activities/isolation & purification , ATPases Associated with Diverse Cellular Activities/metabolism , Cryoelectron Microscopy/methods , DNA/metabolism , Histone Chaperones/isolation & purification , Histone Chaperones/metabolism , Histones/metabolism , Microscopy, Atomic Force , Molecular Dynamics Simulation , Protein Conformation, alpha-Helical , Protein Domains , Protein Structure, Quaternary , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Schizosaccharomyces pombe Proteins/isolation & purification , Schizosaccharomyces pombe Proteins/metabolism , Single Molecule Imaging/methodsABSTRACT
The Rea1 AAA+-ATPase dislodges assembly factors from pre-60S ribosomes upon ATP hydrolysis, thereby driving ribosome biogenesis. Here, we present crystal structures of Rea1-MIDAS, the conserved domain at the tip of the flexible Rea1 tail, alone and in complex with its substrate ligands, the UBL domains of Rsa4 or Ytm1. These complexes have structural similarity to integrin α-subunit domains when bound to extracellular matrix ligands, which for integrin biology is a key determinant for force-bearing cell-cell adhesion. However, the presence of additional motifs equips Rea1-MIDAS for its tasks in ribosome maturation. One loop insert cofunctions as an NLS and to activate the mechanochemical Rea1 cycle, whereas an additional ß-hairpin provides an anchor to hold the ligand UBL domains in place. Our data show the versatility of the MIDAS fold for mechanical force transmission in processes as varied as integrin-mediated cell adhesion and mechanochemical removal of assembly factors from pre-ribosomes.
