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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02463

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Hansenula polymorpha Pex20p is an oligomer that binds the peroxisomal targeting signal 2 (PTS2)

Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, NL-9750 AA Haren, The Netherlands

View larger version (30K): [in a new window]   Fig. 1. PTS2 protein mislocalization in Hppex20 cells. (A) Post nuclear supernatants prepared from glucose/methylamine-grown wild type (WT) and Hppex20 cells were subjected to differential centrifugation. P3-30,000 g pellet, S3-30,000 g supernatant. Western blots were probed with antibodies against several H. polymorpha proteins: AMO (PTS2 matrix protein); porin (mitochondria); ADH-alcohol dehydrogenase (cytosol) and Pex14p (peroxisomal membrane). Equal portions of each fraction were loaded per lane. (B) Fluorescence microscopy of WT and Hppex20 cells expressing a fusion protein consisting of the first 50 amino acids of S. cerevisiae thiolase and GFP and DsRed containing the PTS1 sequence –SKL. In WT cells, green and red fluorescence is present in spots, indicative of peroxisomes. In pex20 cells, green fluorescence is observed in the cytosol, whereas red fluorescence is localized in spots.

View larger version (131K): [in a new window]   Fig. 2. Normal PTS1 protein localization in Hppex20 cells. (A,B) Ultrathin sections of WT and pex20 cells were used for immunolabeling experiments using antibodies against the PTS1 protein alcohol oxidase (A,B) or the PTS2 protein amine oxidase (C,D). Both in WT (A) and in pex20 cells (B) AO labeling is found at the peroxisomal profiles. AMO is normally localized to peroxisomes in WT cells (2C), but mislocalized to the cytosol in pex20 cells (D). Bars, 0.5 µm. M, mitochondrion; N, nucleus, P, peroxisome; V, vacuole.

View larger version (23K): [in a new window]   Fig. 3. HpPex20p is not required for oligomerisation of AMO and thiolase. (A) Western blot analysis of crude cell extracts revealed that the levels AMO and thiolase were similar in WT and Hppex20 cells. Equal amounts of protein were loaded per lane. (B-F) The oligomeric state of AMO (B), thiolase (C), AO (D), catalase (CAT; E) and HpPex20p (F) were determined by gel filtration analysis. Western blotting of FPLC elution fractions of WT and pex20 cell free extracts revealed similar native sizes for AMO (150 kDa; B), thiolase (100 kDa; C), AO (600 kDa; D) and catalase (240 kDa). In crude extracts of WT H. polymorpha cells HpPex20p was estimated to be 180 kDa (F). WT and Hppex20 cells were grown to the late exponential growth phase on glucose/methylamine for the detection of AMO, CAT, AO and HpPex20p (Fig. 3A,B,D-F) or shortly induced on oleic acid/ammonium sulphate to induce thiolase (A,C).

View larger version (20K): [in a new window]   Fig. 4. Levels of HpPex20p. (A) WT and Hppex20 cells were grown on glucose/methylamine medium. Western blots, probed with -HpPex20p antibodies, revealed that in lysates of TCA-precipitated WT cells a cross-reacting protein band with an apparent molecular mass of 35 kDa was present. This band was absent in a similar blot prepared from Hppex20 cells. (B) Crude cell extracts were prepared from TCA-precipitated WT cells grown on glucose (lanes 1,2) or methanol (lanes 3,4) as carbon source in the presence of ammonium sulphate (lanes 1,3) or methylamine (lanes 2,4) as nitrogen source. Western blots, probed with -HpPex20p antibodies, revealed that HpPex20p levels were not significantly increased when cells were grown on media containing methylamine, relative to ammonium sulphate. However, HpPex20p levels appeared to be dependent on the carbon source and higher in glucose grown cells (lanes 1,2) compared with methanol-grown cells (lanes 3,4). (C) Western blot analysis of crude cell extracts prepared from TCA precipitated glucose- (lanes 1-3) and methanol grown (lanes 4-6) H. polymorpha WT cells. Cells were collected prior to (lanes 1,4) or after growth for 60 minutes in the presence (lanes 2,5) or absence (lanes 3,6) of proteasome inhibitor MG-132. Blots were probed with anti-HpPex20p antibodies. Equal amounts of protein were loaded per lane. The addition of MG-132 resulted in an increase in HpPex20p levels during growth of cells on glucose (lane 2), but not on methanol (lane 5).

View larger version (31K): [in a new window]   Fig. 5. Subcellular localization of HpPex20p. (A) Glucose/choline grown WT cells were disrupted using glass beads in buffer lacking (–) or containing (+) a protease inhibitor cocktail and incubated for 10 minutes at room temperature. Crude extracts prepared from cells that were immediately TCA precipitated upon harvesting were used as a control (TCA). HpPex20p levels were analysed by western blotting using anti-Pex20p antibodies. Equal amounts of protein were loaded per lane. These experiments revealed that HpPex20p is partially protected by the addition of the protease inhibitor cocktail (+). However, still approximately 90% is degraded relative to cells that were immediately TCA precipitated. (B) From glucose/choline-grown WT cells a post nuclear supernatant (PNS) was prepared in the presence of the protease inhibitor cocktail and subsequently centrifuged at 30,000 g. Western blot analysis revealed that HpPex20p is present in the organellar pellet. P3-30.000g pellet, S3-30,000 g supernatant. (C) Fluorescence microscopy of WT cells expressing HpPex20.eGFP and DsRed-SKL. This revealed that the green fluorescence was present in a spot at the same localization as the DsRed-SKL fluorescence.

View larger version (16K): [in a new window]   Fig. 6. Examples of experimental autocorrelation curve (dots), fitted (solid line), and residuals (upper inset) of HpPex20p. HpPex20p was labeled with Alexa Fluor 488. In all experiments the concentration of HpPex20p was 50 nM. After global analysis of 20 experimental curves, a diffusion time of 223 microseconds was obtained (confidence limit: 218-277 microseconds). Based on these data the calculated molecular mass of the fluorescent protein complex is estimated to amount 198 kDa (confidence limit: 185-380 microseconds) assuming that the complex is globular in shape.

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