Peroxisomal targeting, import, and assembly of alcohol oxidase in Pichia pastoris

Abstract

Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX-HA). Coexpression of AOX-HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX-HA present in octameric active AOX, suggesting that newly synthesized AOX-HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.

Figure 1

Location of AOX protein in P. pastoris wild-type (WT) and selected pex strains. Methanol-induced cells of each strain were spheroplasted, osmotically lysed, and subjected to differential centrifugation. Protein samples of 30 μg from the resulting organelle pellet (P) and cytosolic supernatant (S) fractions were subjected to SDS-PAGE and immunoblotting with anti-AOX antibodies.

Figure 2

Immunoblot analysis of AOX in organelle pellet (P) and cytosolic supernatant (S) fractions of methanol-induced P. pastoris and H. polymorpha strains expressing heterologous AOX. Group 1 lanes contain 20-μg samples from P. pastoris strain MC200 (pex2Δ aox1Δ aox2Δ) expressing the H. polymorpha MOX gene. Groups 2 and 3 contain 20-μg samples from H. polymorpha strain CW111 (pex10-1 moxΔ) expressing the P. pastoris AOX1 gene at 37° (2) or 30°C (3).

Figure 3

Location of β-lactamase fusion proteins in P. pastoris as determined by immunofluorescence and subcellular fractionation. The photomicrographs contain cells of the strains listed in the left column processed for immunofluorescence using anti-catalase (anti-CAT) and anti–β-lactamase (anti-βLAC) antibodies. The right column shows histograms of the percentage of activity for selected marker enzymes present in crude organelle pellet fractions from the same strains. Cat, Catalase; Lac, β-lactamase; Cyt c ox, Cytochrome c oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Sucrose density gradient of organelle pellet from a methanol-grown P. pastoris strain expressing β-lactamase–LARF. Catalase activity (•) is presented as ΔE240 U/ml; cytochrome c oxidase activity (□) as U/ml × 10⁻², and β-lactamase activity (▾) as U/ml × 10⁻³.

Figure 5

Sucrose density gradient analysis of P. pastoris strains expressing AOX–HA. (A) Total cell extracts (10 μg/lane) prepared from a strain expressing only AOX-WT (lane 1) or AOX-HA (lane 2) and immunoblotted with antibodies against AOX and the HA tag. Sucrose density gradient profiles of strain MC-HWO42 expressing only AOX–HA (B) or strain GS–HWO42 coexpressing both AOX–HA and AOX-WT (C). Symbols in the gradients are: AOX (•), catalase (▾), and cyt c oxidase (□). In B, catalase activities are presented as ΔE240 U/ml, cytochrome c oxidase activities as U/ml, and alcohol oxidase activities as U/ml × 10⁻². In C, catalase activities are presented as ΔE240 U/ml, cytochrome c oxidase activities as U/ml, and alcohol oxidase activities as U/ml × 10⁻¹. The immunoblots shown below each graph contained either 5 (B) or 1 μl (C) of each indicated fraction and were reacted with anti-HA monoclonal antibodies.

Figure 6

Sucrose velocity gradient analysis of AOX–HA expressing P. pastoris strains. Histograms show activity in fractions for tetrameric catalase (∼300 kD; white bars) and octameric AOX (∼600 kD; dark bars) as a percentage of the total activity in the gradient for each enzyme. Immunoblots containing 10-μl samples of gradient fractions were reacted with either anti-AOX or anti-HA antibodies. (A) shows purified active AOX and denatured AOX (AOX*). (B) shows extract prepared from a pex5Δ strain. C–F gradient results from strains expressing the following forms of AOX: C, AOX-WT; D, AOX–RSC; E, AOX–HA; F, both AOX–HA and AOX-WT. Fractions 14–19 from each gradient are not shown since they did not contain significant amounts of protein.

Figure 7

Histogram of two hybrid system assays between Pex5p and the COOH-terminal 22 amino acids of AOX (AOX-WT). Data shown are the mean of three independent experiments. Units indicate β-galactosidase activity expressed as U/mg protein.

Figure 8

Analysis of octameric AOX in the P. pastoris strain coexpressing AOX–HA and AOX-WT. (A) Octameric AOX (fraction 9 from the sucrose velocity gradients shown in Fig. 6) was immunoprecipitated using HA-tag monoclonal antibodies, subjected to SDS-PAGE, and immunoblotted using antibodies against AOX (top) and HA (bottom). The SDS-PAGE lanes were loaded with equal ratios (relative to top and bottom) of sample from the following strains: Lane 1, AOX–HA; lane 2, AOX-WT/AOX–HA; lane 3, nonimmunoprecipitated AOX-WT, and lane 4, AOX-WT. (B) Octameric AOX from fraction 9 of the gradients in Fig. 6 were subjected to electrophoresis on a 5% native (nonreducing, nondenaturing) polyacrylamide gel and immunoblotted using antibodies against AOX (top) and HA (bottom). Lane 1, AOX-WT; lane 2, AOX-WT/AOX-HA; lane 3, AOX–HA; and lane 4, a mixture of fractions from both the AOX–HA strain and the AOX-WT/AOX–HA strain.