Pex17p is required for import of both peroxisome membrane and lumenal proteins and interacts with Pex19p and the peroxisome targeting signal-receptor docking complex in Pichia pastoris

Abstract

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Delta mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Delta mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal-receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.

Figure 1

Fluorescence microscopy of mPTS(Pex3p)-GFP in wild-type and pex mutant cells. Methanol-grown wild-type (PPY12), pex3Δ (SWS3DM), pex19Δ (SKF13), pex1Δ (SWS1DM), and pex8Δ (SWS8DM) strains expressing the mPTS(Pex3p)-GFP were visualized by fluorescence microscopy and Nomarski optics.

Figure 2

FACS analysis of fluorescence intensity of mPTS(Pex3p)-GFP in wild-type and pex3Δ cells. (A) Time course analysis of mPTS(Pex3p)-GFP fluorescence intensity (FL1-H) versus cell number (Counts) from a population of wild-type (SKF1) and pex3Δ (SWS3DM) cells at the indicated times after induction in methanol medium. (B) Mean fluorescence intensity of mPTS(Pex3p)-GFP in the high fluorescence intensity peak of the population plotted versus time.

Figure 2

FACS analysis of fluorescence intensity of mPTS(Pex3p)-GFP in wild-type and pex3Δ cells. (A) Time course analysis of mPTS(Pex3p)-GFP fluorescence intensity (FL1-H) versus cell number (Counts) from a population of wild-type (SKF1) and pex3Δ (SWS3DM) cells at the indicated times after induction in methanol medium. (B) Mean fluorescence intensity of mPTS(Pex3p)-GFP in the high fluorescence intensity peak of the population plotted versus time.

Figure 3

Sequence alignment and features of P. pastoris and S. cerevisiae Pex17p orthologues. The amino acid sequences were aligned using the ClustalW program (A). Identical residues (boxed) and similar residues (gray) are shaded. 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. (B) Sequence features and relative positions. TM, putative transmembrane domain; Coil, putative coiled-coil domain.

Figure 4

Immunofluorescence microscopy of Pex3p in wild-type and pex17Δ cells. Oleate- and methanol-grown wild-type (SMD1163) and pex17Δ (SWS17D) spheroplasts were indirectly labeled with the anti-Pex3p antibody and visualized by fluorescence microscopy and Nomarski optics.

Figure 5

Electron microscopy of wild-type and pex17Δ cells. Methanol-grown wild-type (SMD1163; A and C) and pex17Δ (SWS17D; B and D) cells were prepared as described in MATERIALS AND METHODS for membrane staining (A and B) and immunoelectron microscopy with anti-Pex3p antibodies (C and D). Arrows point to the novel structures in pex17Δ cells (A and B). n, nucleus; m, mitochondria; v, vacuole; p, peroxisome; *, peroxisme remnant.

Figure 6

Mislocalization of peroxisomal proteins by pex17Δ mutants. (A) Oleate-grown spheroplasts of wild-type and pex17Δ cells (SMD1163 and SWS17D) 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 the indicated antibodies. (B) The PNS from oleate-grown cells was adjusted to 65% sucrose, layered with 50 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. Arrows point to the normal, full-length catalase and Pex3p.

Figure 7

Subcellular localization of Pex17HAp. (A) Oleate-grown spheroplasts of pex17Δ cells expressing Pex17HAp (SWS17HA) 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 the indicated antibodies. (B) Methanol-grown spheroplasts of pex17Δ cells expressing Pex17HAp (SWS17HA) were indirectly labeled using anti-Pex3p or anti-HA antibodies as described in MATERIALS AND METHODS and visualized by fluorescence microscopy (Pex3p and Pex17HAp) and Nomarski optics.

Figure 8

Membrane extraction of Pex17HAp. Organelle membranes from oleate-grown cells expressing Pex17HAp (SWS17HA) were extracted with the indicated buffers and repelleted. The supernatant (s) and pellet (p) fractions were analyzed by immunoblotting with the indicated antibodies. carbonate, 0.1 M sodium carbonate, pH 11; Tris, 10 mM Tris-HCl, pH 8.0; buffer, original lysis buffer only; 1% Triton X-100, original lysis buffer with 1% Triton X-100.

Figure 9

Protease sensitivity of Pex17HAp. Organelle pellet fractions from oleate-grown cells expressing Pex17HAp (SWS17HA) were incubated with the indicated amount of trypsin in the presence (+) or absence (−) of Triton X-100. The samples were analyzed by immunoblotting with the indicated antibodies.

Figure 10

Two-hybrid analysis of Pex17p and Pex19p. The indicated Pex17p 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 as described in MATERIALS AND METHODS. Numbers refer to amino acids from Pex19p (A) or Pex17p (B). pAD, transcriptional activation domain fusion constructs; pBD, DNA-binding domain fusion constructs; C, empty DNA-binding domain plasmid (pKNSD55).

Figure 11

Cross-linking and coimmunoprecipitation of Pex17HAp with the PTS–receptor docking complex and Pex19p. Immunoprecipitations from cross-linked (+) or non–cross-linked (−) extracts of the Pex17HAp-expressing strain (SWS17HA) were analyzed by immunoblotting. (A) Pex19p, Pex5p, and Pex7p were immunoprecipitated and immunoblotted with anti-HA. (B) Pex17HAp and Pex14p were immunoprecipitated and immunoblotted with anti-Pex3p. (C) Pex14p was immunoprecipitated and immunoblotted with anti-Pex19p. (D) Pex19p, Pex5p, and Pex7p were immunoprecipitated and immunoblotted with anti-Pex3p. Whole-cell lysates (wc) were loaded (0.033 A₆₀₀) as a control for immunoblotting.

Figure 12

Schematic representation of various putative Pex protein subcomplexes. The rationale for the definition of these complexes is explained in DISCUSSION. The question mark by the arrows represents a putative bridged interaction, which is not proven or disproven by the data. Because complex formation may occur on the membrane or in the cytosol, no membrane bilayer was included in the diagram for A, B, and D.