In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p.

Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisomedeficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14). The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N-and Cterminal parts to the cytosol. Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40 -42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

Thin films of the ferromagnetic metal SrRuO3 (SRO) show a varying easy magnetization axis depending on the epitaxial strain and undergo a metal-to-insulator transition with decreasing film thickness. We have investigated the magnetic properties of SRO thin films with varying thicknesses fabricated on SrTiO3(001) substrates by soft x-ray magnetic circular dichroism (XMCD) at the Ru M2,3 edge. Results have shown that, with decreasing film thickness, the film changes from ferromagnetic to non-magnetic around 3 monolayer thickness, consistent with previous magnetization and magneto-optical Kerr effect measurements. The orbital magnetic moment perpendicular to the film was found to be ∼ 0.1 µB/Ru atom, and remained nearly unchanged with decreasing film thickness while the spin magnetic moment decreases. Mechanism for the formation of the orbital magnetic moment is discussed based on the electronic structure of the compressively strained SRO film.

The peroxisome targeting signal type 1 (PTS1) receptor, Pex5p, of the tetratricopeptide repeat (TPR) motif family is located mostly in the cytosol and mediates the translocation of PTS1 proteins to peroxisomes. As a step towards understanding the mechanisms of protein import into peroxisomes, we investigated the molecular mechanisms involved in PTS1 recognition by Pex5p with regard to requirement of energy and cytosolic factors, using cell-free synthesized acyl-CoA oxidase (AOx) as a PTS1 cargo protein, together with Pex5p and heat-shock protein (Hsp)70 from rat liver. Pex5p was partly associated with peroxisomes of rat liver, was resistant to washing with a high concentration of salt and to alkaline extraction and was inaccessible to protease added externally. Pex5p bound to AOx in an ATP-dependent manner. AOx synthesized in a cell-free translating system from rabbit reticulocyte lysate was imported into peroxisomes without being supplemented with Pex5p and Hsp70, implying that peroxisome-associated Pex5p was released from the membranes and functional in this in vitro import assay. Antibodies against Pex5p and Hsp70 inhibited AOx import. In contrast, AOx synthesized in a wheat-germ lysate required the external addition of Pex5p for import, in which Hsp70 augmented the AOx import. The TPR domain of Pex5p was revealed to bind to the N-terminal part in an Hsp70-independent manner, whereas mutual interaction of the TPR region was noted in the presence of Hsp70. Hsp70 interacted with the TPR domain of Pex5p. Moreover, Hsp70 and ATP synergistically enhanced the binding of Pex5p to the C-terminal PTS1-containing part of AOx, implying that Pex5p recognizes its cargo PTS1 protein by chaperone-assisted as well as energy-dependent mechanisms in vivo.

The intracellular distribution of a membrane-bound protein disulfide isomerase (PDI) in rat liver was studied by quantitative immunoprecipitation, and its microsomal localization was confirmed. The content of the enzyme was 1 to 2% of total microsomal protein, and it was almost equally distributed between rough and smooth microsomes. The enzyme was not solubilized from microsomes by high concentrations of KCl, but was readily solubilized by detergents. Since PDI in microsomes was susceptible to digestion by trypsin, at least some parts of the enzyme molecule are exposed on the outside surface of microsomal vesicles. However, the binding of antibodies to microsomal PDI and the modification of the glutamine residues of PDI molecules by transglutaminase suggested that the molecules are not extensively exposed on the surface. Solubilized PDI was unable to rebind to microsomes or to become incorporated into reconstituted membrane of detergent-solubilized microsomes, showing that the association of this enzyme with the membrane is not simply mediated by hydrophobic interaction.

Magnetic anisotropies of ferromagnetic thin films are induced by epitaxial strain from the substrate via strain-induced anisotropy in the orbital magnetic moment and that in the spatial distribution of spin-polarized electrons. However, the preferential orbital occupation in ferromagnetic metallic La 1−x Sr x MnO 3 (LSMO) thin films studied by x-ray linear dichroism (XLD) has always been found out-of-plane for both tensile and compressive epitaxial strain and hence irrespective of the magnetic anisotropy. In order to resolve this mystery, we directly probed the preferential orbital occupation of spin-polarized electrons in LSMO thin films under strain by angle-dependent x-ray magnetic circular dichroism (XMCD). Anisotropy of the spin-density distribution was found to be in-plane for the tensile strain and out-of-plane for the compressive strain, consistent with the observed magnetic anisotropy. The ubiquitous out-of-plane preferential orbital occupation seen by XLD is attributed to the occupation of both spin-up and spin-down out-ofplane orbitals in the surface magnetic dead layer.

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