Protein import into peroxisomes depends on a complex and dynamic network of protein-protein interactions. Pex14 is a central component of the peroxisomal import machinery and binds the soluble receptors Pex5 and Pex19, which have important function in the assembly of peroxisome matrix and membrane, respectively. We show that the N-terminal domain of Pex14, Pex14(N), adopts a three-helical fold. Pex5 and Pex19 ligand helices bind competitively to the same surface in Pex14(N) albeit with opposite directionality. The molecular recognition involves conserved aromatic side chains in the Pex5 WxxxF/Y motif and a newly identified F/YFxxxF sequence in Pex19. The Pex14-Pex5 complex structure reveals molecular details for a critical interaction in docking Pex5 to the peroxisomal membrane. We show that mutations of Pex14 residues located in the Pex5/Pex19 binding region disrupt Pex5 and/or Pex19 binding in vitro. The corresponding full-length Pex14 variants are impaired in peroxisomal membrane localisation in vivo, showing that the molecular interactions mediated by the N-terminal domain modulate peroxisomal targeting of Pex14.
One of the earliest steps in metazoan pre-mRNA splicing involves binding of U2 snRNP auxiliary factor (U2AF) 65 KDa subunit to the polypyrimidine (Py) tract and of the 35 KDa subunit to the invariant AG dinucleotide at the intron 3' end. Here we use in vitro and in vivo depletion, as well as reconstitution assays using purified components, to identify hnRNP A1 as an RNA binding protein that allows U2AF to discriminate between pyrimidine-rich RNA sequences followed or not by a 3' splice site AG. Biochemical and NMR data indicate that hnRNP A1 forms a ternary complex with the U2AF heterodimer on AG-containing/uridine-rich RNAs, while it displaces U2AF from non-AG-containing/uridine-rich RNAs, an activity that requires the glycine-rich domain of hnRNP A1. Consistent with the functional relevance of this activity for splicing, proofreading assays reveal a role for hnRNP A1 in U2AF-mediated recruitment of U2 snRNP to the pre-mRNA.
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