Pex19p interacts with Pex3p and Pex10p and is essential for peroxisome biogenesis in Pichia pastoris

Abstract

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Delta cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.

Figure 1

Sequence alignment of Pex19p orthologues. The amino acid sequences were aligned using the ClustalW program. Identical residues (black) and similar residues (gray) in at least half of the orthologues are shaded. Pp, P. pastoris Pex19p; Sc, S. cerevisiae Pex19p; Hs, Homo sapiens HK33/PxP; Cg, Cricetulus griseus PxP; Sp, S. pombe hypothetical protein C17C9.14; Ce, C. elegans hypothetical protein F54F2.8. Similarity rules: G = A = S, A = V, V = I = L = M, I = L = M = F = Y = W, K = R = H, D = E = Q = N, and S = T = Q = N. Dashes represent gaps.

Figure 2

pex19Δ mutants are defective for the import of catalase (PTS1) and PTS2-GFP. Wild-type and pex19Δ cells (SMD1163 and SKF14) grown in methanol media were prepared for anti-catalase immunofluorescence, and PTS2-GFP expressing wild-type and pex19Δ cells (STW2 and AK11) grown in oleate media were visualized using a fluorescence microscope (anti-catalase, GFP) and Nomarski optics as described in MATERIALS AND METHODS.

Figure 3

Steady-state levels of peroxisomal proteins during oleate and methanol growth of wild-type and pex19Δ cells. Glucose-grown wild-type and pex19Δ cells (PPY12 and SKF13) were shifted to oleate (A) or methanol media (B), and samples were collected at the indicated times and processed as described in MATERIALS AND METHODS for SDS-PAGE and immunoblotting with the indicated antibodies.

Figure 4

Mislocalization of peroxisomal proteins by pex19Δ mutants. Oleate-grown (A) and methanol-grown (B) spheroplasts of wild-type and pex19Δ cells (SKF14 and SMD1163) were lysed and subjected to sequential differential centrifugation. Equivalent amount of the PNS, 27,000 × g supernatant (S27), 27,000 × g pellet (P27), 100,000 × g supernatant (S100), and 100,000 × g pellet (P100) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antibodies. (C) The PNS from methanol-grown cells was adjusted to 60% sucrose, layered with 55 and 35% sucrose, and centrifuged to allow floatation of membranes into the lighter fractions as described in MATERIALS AND METHODS. P, pelleted material at the bottom of the tube. Fractions are numbered from the bottom to the top and were collected as described in MATERIALS AND METHODS. Fractions 1 and 2 are the 60% sucrose load zone; fractions 3 and 4 are the 55% sucrose float zone. The top fraction contained no protein.

Figure 5

Immunofluorescence microscopy of Pex3p-containing compartments in wild-type and pex mutant cells. Methanol-grown wild-type (PPY12), pex19Δ (SKF13), pex2Δ (PPY12Δ2), and pex5Δ (PPY12Δ5) spheroplasts were indirectly labeled with the anti-Pex3p antibody and visualized by fluorescence microscopy (Pex3p) and Nomarski optics.

Figure 6

Deconvolution microscopy of Pex3p immunofluorescence from wild-type and pex19Δ cells. Pex3p-containing compartments from wild-type (PPY12) (A) and pex19Δ (SKF13) (B) cells were visualized by immunofluorescence staining with Pex3p antibodies on a Delta Vision optical sectioning microscope. Three consecutive 0.2-μm sections are shown from each strain. Bar, 2 μm.

Figure 7

Electron microscopy of wild-type and pex19Δ cells. Methanol-grown wild-type (PPY12) (A) and pex19Δ (SKF13) (B) cells were prepared as described in MATERIALS AND METHODS. (C) Enlargement of the area highlighted by dashed white box in B. n, nucleus; m, mitochondria; v, vacuole; p, peroxisome.

Figure 8

Induction and subcellular fractionation of Pex19p. (A) Glucose-grown wild-type cells (PPY12) were shifted to oleate and methanol media, and samples were collected at the indicated times and processed as described for Figure 3 for immunoblotting with anti-Pex19p antibodies. (B) Oleate-grown spheroplasts of wild-type and pex19Δ cells (SMD1163 and SKF14) were lysed and subjected to sequential differential centrifugation. Equivalent amounts of the PNS, 27,000 × g supernatant (S27), 27,000 × g pellet (P27), 100,000 × g supernatant (S100), and 100,000 × g pellet (P100) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-Pex19p antibodies.

Figure 9

The putative farnesylation site is not essential for Pex19p function. (A) Cells were grown on plates supplemented with methanol (left panel) or oleate (right panel) as sole carbon source. (B) Indirect immunofluorescence for catalase in methanol-grown pex19Δ cells and pex19Δ cells transformed with the indicated Pex19p-expressing plasmids. (C) Western blot analysis of total extracts of methanol-grown cells probed with Pex3p antibodies. PEX19, wild-type (SMD1163); pex19Δ, deletion mutant (SKF14); pex19Δ + WT, deletion mutant transformed with the wild-type PEX19 (WSP50); pex19Δ + C296S, deletion mutant transformed with the pex19C296S allele (WSP51); pex19Δ + ΔC4, deletion mutant transformed with the pex19C296ter allele (WSP52).

Figure 10

Two-hybrid analysis of Pex3p and Pex19p. The indicated Pex3p and Pex19p hybrid protein constructs were tested for trans-activation of the HIS3 gene, resulting in growth on media lacking histidine (+) or LacZ, resulting in the production of β-galactosidase assayed by blue color change (+) as described in MATERIALS AND METHODS. Numbers refer to amino acids from Pex3p (A) or Pex19p (B). TM, relative position of the putative transmembrane domain in Pex3p.

Figure 11

Two-hybrid analysis of Pex10p and Pex19p. The indicated Pex10p and Pex19p hybrid protein constructs were tested for trans-activation of the HIS3 gene, resulting in growth on media lacking histidine (+) or LacZ, resulting in the production of β-galactosidase assayed by blue color change (+) as described in MATERIALS AND METHODS. Numbers refer to amino acids from Pex10p (A) or Pex19p (B). Zn, relative position of the zinc-binding domain in Pex10p.

Figure 12

Coimmunoprecipitation of Pex3p and NH-Pex10p with Pex19p. Pex19p was immunoprecipitated from cleared lysates of wild-type (SMD1163), pex19Δ (SKF14), or NH-PEX10 (SSH18) strains as described in MATERIALS AND METHODS. The material was resolved by SDS-PAGE and analyzed by immunoblotting with anti-Pex19p (A), anti-Pex3p (B), and anti-NH antibodies (C).