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Identification and characterization of a novel human plant pathogenesis-related protein that localizes to lipid-enriched microdomains in the Golgi complex

¹ Biochemie-Zentrum Heidelberg (BZH), University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
² Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany
³ Central Spectroscopy Department, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
⁴ Max-Planck Institut für Biochemie, Martinsried, Germany

View larger version (72K): [in a new window]   Fig. 1. Protein sequence and alignment of GAPR-1. (A) Peptide sequences determined by microsequencing. (B) cDNA-derived sequence of GAPR-1. The dotted lines indicate the position of the peptides described in (A). The consensus sequence for N-myristoylation is indicated by the box, and the potential protein-protein interacting sites (coiled-coil region and caveolin-interacting region) are marked in grey. (C) Amino-acid sequence alignment of GAPR-1 with relatives of the superfamily. For each of the subfamilies, including human GliPR, CRISPs, plant PR-1 proteins, allergens of insect venoms and snake or lizard venoms, the protein sequence of the member with the highest homology to GAPR-1 is aligned to GAPR-1. Other sequences shown are (with Swiss Prot accession numbers between brackets) Drosophila CG2337 (AE003674), pathogenesis-related protein PR1 (U64806), hypothetical 30.6 kDa protein in yeast PRY1 (P47032), venom allergen 5 AG5 (P35784), testis-specific protein TPX1 (P16562) and Glioma pathogenesis-related protein GliPR (P48060). Identical amino-acid residues in family members are highlighted in black. Conservative amino-acid exchanges are highlighted in grey.

View larger version (22K): [in a new window]   Fig. 2. GAPR-1 is a Golgi-localized peripheral membrane protein. (A) The enrichment of GAPR-1 in CHO Golgi membranes and CHO GIC was determined by comparing the amounts of GAPR-1 in CHO homogenate (lane 1, 50 µg), CHO cytosol (lane 2, 50 µg), CHO Golgi membranes (lane 3, 5 µg) and CHO GIC (lane 4, 0.5 µg) by use of SDS-PAGE and western blotting. (B) The topology of GAPR-1 at Golgi membranes was determined by protease digestion of Golgi membranes (lanes 2-4) in the absence or presence of Trypsin inhibitor (lane 3) or Triton X-100 (lane 4). After incubation (as described in Materials and Methods), the proteins were separated by SDS-PAGE and GAPR-1 and p23 were visualized by western-blotting. (C) Exclusion of GAPR-1 from COPI-coated vesicles. Equivalent amounts of isolated Golgi membranes and COPI-coated vesicles (10.8 µg total phospholipid) were analysed by SDS-PAGE and western blotting for the presence of GAPR-1. As positive controls for COPI-coated vesicles, the blots were probed with antibodies against ß-COP and p23. Golgi membranes and COPI-coated vesicles were isolated as described in Materials and Methods.

View larger version (30K): [in a new window]   Fig. 4. Strong interaction of GAPR-1 with Golgi membranes. In all panels, the incubations were analysed by SDS-PAGE and western blotting for the presence of the indicated proteins. (A) CHO cells were incubated for 30 minutes in the absence (lane 2 and 3) or presence of 5 µM Brefeldin A (lane 4 and 5). The BFA-induced redistribution of GAPR-1 into tubulo-vesicular structures was confirmed by concomitant immunofluorescence (data not shown). After homogenisation, the homogenate was centrifuged for 1hour at 100,000 g to yield a total membrane (lanes 2 and 4) and cytosolic fraction (lanes 3 and 5). GAPR-1 was immunoprecipitated from the membrane fraction (2 mg) or from the cytosolic fraction (2 mg) as described in the Materials and Methods. As a control, GAPR-1 was immunoprecipitated from isolated CHO Golgi membranes (50 µg) (lane 1). (B) 50 µg of CHO Golgi membranes (lanes 1-3) was incubated with 1 M KCl (lane 2) or with 0.1 M Na2CO3, pH 11 (lane 3) for 30 minutes on ice. After centrifugation through a 15% (w/v) sucrose cushion, equal amounts of membrane (29 nmol phospholipid) were analysed. (C) CHO Golgi membranes (25 µg) were incubated for 30 minues at 4°C in the absence (lane 1) or presence (lanes 2 an 3) of 3 µl of bacterially expressed, purified and non-myristoylated GAPR-1 (5.3 mg/ml) in 25 mM Hepes/KOH, pH 7.2, 20 mM Kcl, 2.5 mM magnesium acetate, 0.1 M sucrose, 1 mg/ml ovalbumine and 10 mM DTT. KCl (1 M final concentration) was added to one incubation (lane 3). Golgi membranes were re-isolated by centrifugation through a 15% (w/v) sucrose cushion. (D) CHO Golgi membranes (50 µg) were incubated with Hydroxylsulfosuccinimidyl-4-azidobenzoate (5 mM) in PBS for 30 minutes at RT and left on ice (lane 1) or irradiated for 10 minutes at 254 nm (lane 2 and 3) and analysed for crosslinked products. For immunoprecipitation (lane 3), 500 µg of Golgi membranes was used, and GAPR-1 was immunoprecipitated as described in Materials and Methods.

View larger version (22K): [in a new window]   Fig. 3. GAPR-1 is myristoylated in vivo. (A) Positive nanoESI spectrum of the in-gel digest of native GAPR-1. The top panel shows the complete survey spectrum, and the lower panel shows the expanded view from m/z 413 to 417 showing the singly protonated molecular ion of the T1 fragment including the 13C isotope peak. (B) Positive nanoESI product ion spectrum of m/z 414.33. The spectrum shows the key fragments for the myrG structure at m/z 211, 240 and 268 and sequence-specific fragment ions, which identify the peptide as the T1 fragment myrGK of GAPR-1.

View larger version (86K): [in a new window]   Fig. 5. Immunolocalization of GAPR-1 to the Golgi complex. Vero cells were incubated in the absence (top panels) or presence of BFA (5mM) for the indicated times (5-30 minutes) and processed for double immunofluorescence. Left and middle panels represent immunofluorescent labelling of the cells with an antibody against GAPR-1 and the KDEL receptor, respectively. The right panels shows the colocalization of GAPR-1 and KDEL receptor by merging the two panels. Bar, 20µm.

View larger version (30K): [in a new window]   Fig. 6. Colocalization of GAPR-1 with anterograde cargo in the intermediate compartment. Vero cells were infected with vesicular stomatitis virus tsO45 (VSV-G tsO45) expressing a glycoprotein with temperature-sensitive folding properties. After infection, the cells were incubated at the non-permissive temperature of 39.5°C (upper panels). Two representative examples are shown of the subsequent incubation of the cells at 15°C (middle and lower panels). The cells were analysed for the localization of GAPR-1 and VSV-G by double immunofluorescence as described in the Materials and Methods. Bar, 20 µm.

View larger version (23K): [in a new window]   Fig. 7. Detergent-insolubility of GAPR-1 is not sensitive to BFA. NRK cells were incubated for 15 minutes at 37°C in the absence or presence of BFA (5 µM). The BFA-induced redistribution of GAPR-1 into tubulo-vesicular structures was confirmed by concomitant immunofluorescence (data not shown). After incubation, the cells were directly solubilized in PEN+1% TX-100 and incubated for 30 minutes at 0°C. The lysate was analysed for the presence or absence of low-density detergent insoluble complexes by isopycnic sucrose density centrifugation as described in the Materials and Methods. The gradients were fractionated and the fractions analysed by SDS-PAGE and western blotting for the presence of GAPR-1.

View larger version (33K): [in a new window]   Fig. 8. Differential expression of GAPR-1. After homogenization of the various tissues and preparation of a postnuclear fraction, a total membrane fraction was prepared, and 100 µg of each membrane fraction was analyzed for the presence of GAPR-1 by SDS-PAGE and western blotting. All membrane fractions were isolated from rat tissues, except monocytes, which were isolated from human blood. As a positive control, CHO GIC (0.5 µg) was loaded in lane 16.

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