Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly

Abstract

At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein.

Figure 1

Restoration of peroxisomes in CG-J CHO mutant cells. (a) Peroxisome-deficient mutant ZP119 cells. (b) Peroxisome-restored ZP119, after lipofection with a combined pool (F6-19) of human cDNA library. Arrows indicate the complemented cells. Cytosolic appearance of catalase was apparent in the other cells. (c and d) 119P19 cells, stable HsPEX19-transformants of ZP119 cells. Cells were stained with antisera to catalase (a and b), PTS1 peptide (c), and PMP70 (d), respectively. (Magnification: ×630; bar = 20 μm.)

Figure 2

Amino acid sequence alignment of PEX19 protein from two mammalian species and S. cerevisiae Pex19p. Deduced amino acid sequence of human (Hs) PEX19 was compared with those of Pex19p from Chinese hamster (Cl) and S. cerevisiae (Sc). -, a space. Identical amino acids between species, including two mammalian ones, are shaded, and the CAAX motif is boxed. The sequence used for chemical synthesis of Pex19p peptide is underlined. The solid arrowhead indicates the position of mutation in CG-J PBD patient (see Fig. 4). The database accession numbers for the human PEX19 cDNA is AB018541.

Figure 3

Complementation of biogenesis of peroxisomal enzymes. (A) Latency of catalase activity in CHO-K1, ZP119, and 119P19 cells. □, CHO-K1; ●, ZP119, ○, 119P19; ▴, lactate dehydrogenase in ZP119. Relative free enzyme activity is expressed as a percentage of the total activity measured in the presence of 1% Triton X-100 (5). The results represent a mean of duplicate assays. (B) Biogenesis of peroxisomal proteins. Cell lysates (≈1.8 × 10⁵ cells) were subjected to SDS/PAGE and transferred to polyvinylidene difluoride membrane. Cell types are indicated at the top. Immunoblotting was done with antibodies to acyl-CoA oxidase and thiolase. Arrowheads show the positions of acyl-CoA oxidase components, A, B and C; open and solid arrowheads indicate a larger precursor (P) and mature protein (M) of thiolase, respectively. Dots indicate nonspecific bands (14).

Figure 4

Complementation of fibroblasts from a CG-J Zellweger patient. (A) Partial sequence and deduced amino acid sequence of PEX19 cDNA isolated from a normal control (Left) and a ZS patient PBDJ-01 (Center) are shown. PCR was also done for DNA from PBDJ-01 fibroblasts (Right). One-base insertion A⁷⁶⁴ (shaded), in a codon for Met²⁵⁵, causes a frameshift in PBDJ-01 PEX19 sequence. (B) Sequence comparison of the C-terminal part of Pex19p each from a control and a patient PBDJ-01. Amino acid sequence resulted from the frameshift by the 1-bp insertion in PBDJ-01 is underlined. The arrowhead indicates the position of frameshift mutation. The CAAX box is double-underlined. (C) Transfection of PEX19 from a normal control and a CG-J patient (PBDJ-01). (a) PBDJ-01 fibroblasts. (b) PBDJ-01 fibroblasts were transfected with pUcD2Hyg⋅HsPEX19. (c and d) PBDJ-01 fibroblasts were transfected with PBDJ-01-derived PEX19 cDNA, PEX19A764ins, and a mutant PEX19, PEX19C296S, respectively. (e and f) flag-tagged PEX19C296S, flag-PEX19C296S, was expressed in ZP119. Cells were stained with antibodies to human catalase (a–d), rat catalase (e), and flag (f). Note that peroxisomes were restored only in b, but not in c–f. (Bar = 20 μm.)

Figure 5

Farnesylation of Pex19p. Size comparison of in vitro transcription/translation product of normal and mutated PEX19 cDNA and Pex19p of CHO cells. In vitro transcription/translation product of HsPEX19 and CHO-K1 cell-lysates were subjected to SDS/PAGE. Immunodetection was done for lane 1, as in Fig. 3B, by using anti-Pex19p antibody; radioactive bands were detected by a FujiX BAS1500 Bio-Imaging Analyzer at exposures for 16 h (lanes 2–4, 7, and 8) and 72 h (lanes 5 and 6). Lanes: 1, 40 μg of CHO-K1 cell-lysates; 2, in vitro transcription/translation product (1 μl) of HsPEX19 in the presence of [³⁵S]methionine and [³⁵S]cysteine as label; 3 and 4, immunoprecipitation of ³⁵S-Pex19p (3.5 μl) was done with preimmune and anti-Pex19p immune sera, respectively; 5 and 6, in vitro transcription/translation product (15 μl) of HsPEX19 and HsPEX19C296S, respectively, using [³H]farnesyl pyrophosphate as label; 7 and 8, total (1 μl) and immunoprecipitate (3.5 μl) from in vitro transcription/translation product of HsPEX19C296S using [³⁵S]methionine/cysteine. Solid and open arrowheads indicate farnesylated and nonfarnesylated Pex19p, respectively (see text).

Figure 6

Intracellular localization and topology of Pex19p. N-terminally flag-tagged human Pex19p was expressed in CHO-K1 cells. Cells were treated with 0.1% Triton X-100 (a and b), or with 25 μg/ml of digitonin, under which the plasma membrane was permeabilized (17, 18) (c and d). Cells were stained with antibodies to flag (a and c) and PTS1 (b and d). Note that punctate structures, peroxisomes, are superimposable in a and b and that flag-Pex19p was detected after both types of treatments (a and c). A diffuse staining pattern was partly detected in a and c (see text). (Bar = 20 μm.)

Figure 7

Kinetics of peroxisome biogenesis. (A) ZP119EG1 cells expressing EGFP-PTS1 were transfected with pUcD2Hyg⋅HsPEX19, then monitored by fluorescent microscope. (a–c) PMP70 was visualized by using rabbit anti-PMP70 antibody and Texas Red-labeled goat anti-rabbit IgG antibody. (d–f) EGFP-PTS1. (g–i) Catalase in other cells detected by anti-catalase antibody, as for PMP70. (a, d, and g) 10 h after transfection, (b, e, and h) 16 h. (c, f, and i), 25 h. Note that PMP70, but not EGFP-PTS1 and catalase, is already in numerous vesicular structures at 10 h (see text). (Bar = 20 μm.) (B) Expression of human Pex19p in ZP119. Pex19p was detected by immunoblotting HsPEX19-transfected ZP119 lysates (1.3 × 10⁵ cells at 0 h), at indicated time, where cell-doubling time was 22 h. Arrowhead, Pex19p.