Alopecia in a novel mouse model RCO3 is caused by mK6irs1 deficiency.

Reduced coat 3 (Rco3) is a new spontaneous autosomal recessive mutation with defects in hair structure and progressive alopecia. Here we describe chromosomal mapping and molecular identification of the Rco3 mutation. The murine Rco3 locus maps to a 2-Mb interval on chromosome 15 encompassing the keratin type II gene cluster. Recently, mK6irs1 was described as a type II keratin expressed in Henle's and Huxley's layer of the murine inner root sheath. Genomic sequencing revealed a 10-bp deletion in exon 1 of mK6irs1 resulting in a frameshift after 58 amino acid residues and, therefore, the absence of 422 carboxy-terminal amino acid residues containing the complete alpha-helical rod domain. Henle's and Huxley's layers show no immunoreactivity with mK6irs1-specific antibodies and the absence of intermediate filament formation in electron microscopic images. These results indicate that the expression of functional mK6irs1 is indispensable for intermediate filament formation in the inner root sheath and highlights the importance of the keratinization of the inner root sheath in the normal formation of the hair shaft.

Expression of peroxisomal proteins provides clear evidence for the presence of peroxisomes in the male germ cell line GC1spg.

Peroxisomes are cell organelles that perform multiple functions in the metabolism of lipids and of reactive oxygen species. They are present in most eukaryotic cells. However, they are believed to be absent in spermatozoa and they have never been described in male germ cells. We have used the immortalized germ cell line GC1spg to investigate the expression of peroxisomal proteins in germ cells of mice. The GC1spg cells represent the differentiation state of type B spermatogonia or preleptotene spermatocytes. We could show that peroxisomal membrane proteins like Pmp70 and Pex14p as well as peroxisomal matrix proteins like catalase or acyl CoA oxidase are expressed in GC1spg cells. All these proteins were colocalized in the same structures within the cells. Furthermore, by electron microscopy we have identified subcellular particles with an ultrastructural appearance that is characteristic of peroxisomes. This is the first report demonstrating the peroxisomal compartment in male germ cells of mice.

Genomic organization, chromosomal localization and tissue specific expression of the murine Pxmp2 gene encoding the 22 kDa peroxisomal membrane protein (Pmp22).

Peroxisomes are subcellular organelles with important functions in lipid metabolism that are found in virtually all eucaryotic cells. The peroxisomal membrane contains a number of integral and peripheral membrane proteins involved in the import of peroxisomal matrix proteins and the transport of metabolites across the membrane. The most abundant peroxisomal membrane protein (Pmp) in rat peroxisomes is Pmp22, a 22 kDa protein of unknown function that is encoded by the Pxmp2 gene. To investigate the function of the Pxmp2 gene, we have initiated mouse knockout studies. The expression level of the Pxmp2 mRNA in mice was investigated by Northern blot analysis. Pxmp2 RNA was shown to be differentially expressed with highest expression levels in liver, kidney and in heart tissue. Comparison with other peroxisomal marker genes revealed that the expression of Pxmp2, Pmp70 (Pxmp1) and catalase was regulated independently. Using 5' and 3' RACE we have cloned the full-length cDNA of murine Pxmp2 which comprises 863 nucleotides and have isolated a genomic clone containing the entire murine Pxmp2. We have analyzed the complete intron/exon structure of the Pxmp2 gene which contains five exons spanning about 11 kb on the genomic clone. All intron/exon splice junctions conform to the GT/AG rule. Sequence analysis of the Pxmp2 5' flanking region revealed that it was devoid of a TATA box, but characteristic promoter elements were identified within 250 base pairs upstream of the transcriptional start site. Using a mouse/hamster radiation hybrid panel, Pxmp2 was localized on mouse chromosome 5 at 59 cM.

Reporter-linked monitoring of transgene expression in living cells using the ecdysone-inducible promoter system.

Inducible promoter systems such as the ecdysone-inducible system or the tetracycline-regulated expression systems have proven to be powerful tools in studying gene function. In practice, such systems have met with the difficulty that either the vector expressing the transactivator gene or the vector carrying the response element are frequently silenced by flanking genomic sequences after stable integration. In order to identify those cells in a heterogeneous population in which a transgene is expressed from an ecdysone-inducible promoter, we have created the vector p2ER-EGFP/mcs that contains two ecdysone-inducible expression cassettes in tandem. Using two reporter genes, lacZ and green fluorescent protein (EGFP), we demonstrate that the expression of both genes can be co-induced from a very low baseline in CHO cells expressing the modified ecdysone receptor and the retinoid X receptor. The expression of EGFP and lacZ from vector p2ER-EGFP/lacZ follows the same Muristerone A concentration-dependence as that of EGFP from vector pER-EGFP, indicating that the juxtaposition of the two inducible promoters in vector p2ER-EGFP/mcs does not cause cross interference between them. We suggest that this modification of the ecdysone-inducible promoter system will allow for the visual control of the induced expression of other genes by Muristerone A.

Analysis of the peroxisomal acyl-CoA oxidase gene product from Pichia pastoris and determination of its targeting signal.

Acyl-CoA oxidase (Pox1p) is involved in the beta-oxidation of fatty acids and is targeted to the peroxisomal matrix via the use of different signals in various organisms. In rat, mouse and human, Pox1p contains a canonical peroxisomal targeting signal 1 (PTS1), whereas in the yeasts Candida tropicalis, Saccharomyces cerevisiae, C. maltosa and Yarrowia lipolytica neither a PTS1 nor a PTS2 sequence is present, suggesting that Pox1p might be targeted to the peroxisomes via a third unknown pathway. Alternatively, since proteins lacking a PTS sequence can enter peroxisomes in association with other polypeptides containing a PTS, Pox1p might 'piggy-back' its way into the peroxisomal matrix together with other proteins. To understand the mechanism of peroxisomal targeting of a yeast Pox1p, we cloned the Pichia pastoris POX1 gene to study the pathway of import of PpPox1p into peroxisomes. The gene was cloned by PCR, hybridization and plasmid rescue. The 2157 bp gene encodes a protein with a predicted molecular weight of 80 kDa. Antisera against PpPox1p detected a protein specifically induced on oleate with an apparent molecular weight of 72 kDa. Immunolocalization studies confirmed the peroxisomal localization of PpPox1p. The carboxy-terminus of PpPox1p ends with a PTS1-like sequence, APKI. The sequence PKI was necessary for transport of PpPox1p into peroxisomes and interacted with the PTS1 receptor, Pex5p. Furthermore, addition of the sequence APKI to the C-terminus of the green fluorescent protein directed this fusion protein to the peroxisome. Therefore, PpPox1p uses the PTS1 pathway for its import into peroxisomes.

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

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.

The Pichia pastoris dihydroxyacetone kinase is a PTS1-containing, but cytosolic, protein that is essential for growth on methanol.

Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts. We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that encodes a 65.3 kDa protein with high homology to DAK from Saccharomyces cerevisiae. Although DAK from P. pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic. The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dak delta mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dak delta mutant and the import of peroxisomal proteins was not disturbed. The dak delta mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dak delta mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate. The metabolic pathway explaining the reduced growth rate of the dak delta mutant on dihydroxyacetone is discussed. The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198.

Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting.

Immuno-isolation is a powerful technique for the isolation of cells as well as subcellular organelle populations based on their antigenic properties. We have established a method for immuno-isolation of peroxisomes (PO) from both rat liver and the human hepatoblastoma cell line HepG2 using magnetic beads as solid support. A polyclonal antibody raised against the cytoplasmic C-terminal 10 amino acids of the rat 70 kDa peroxisomal membrane protein was covalently bound to magnetic beads (Dynabeads M-450). The coated beads were incubated with a light mitochondrial fraction and the organelle-bead complexes formed were separated by magnetic sorting in a free-flow system without pelleting the complexes during the isolation procedure. Scanning electron microscopy revealed decoration of beads with particles measuring 150-400 nm in diameter. The particles were identified as PO by catalase cytochemistry and biochemically by marker enzyme analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as immunoblotting for specific detection of peroxisomal matrix, core and membrane proteins. The functional significance of PO in man is emphasized by the existence of inherited diseases such as the Zellweger syndrome in which intact PO are lacking, but peroxisomal remnants called "ghosts" are observed instead. Peroxisomal disorders are usually studied using skin fibroblast cell lines derived from afflicted patients and immuno-magnetic separation may prove particularly useful for the investigation of such cultured cells and for further elucidation of the pathogenesis of fatal peroxisomal disorders.

Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris.

The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis. We have cloned the PpPAS2 gene by functional complementation and show that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa. In a Pppas2 null mutant, import of both peroxisomal targeting signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractionation and fluorescence microscopy. No morphologically distinguishable peroxisomal structures could be detected by electron microscopy in Pppas2 null cells induced on methanol and oleate, suggesting that PpPas2p is involved in the early stages of peroxisome biogenesis. PpPas2p is a peroxisomal membrane protein (PMP) and is resistant to extraction by 1 M NaCl or alkaline sodium carbonate, suggesting that it is a peroxisomal integral membrane protein. Two hydrophobic domains can be distinguished which may be involved in anchoring PpPas2p to the peroxisomal membrane. PpPas2p is homologous to the Saccharomyces cerevisiae Pas3p. The first 40 amino acids of PpPas2p, devoid of the hydrophobic domains, are sufficient to target a soluble fluorescent reporter protein to the peroxisomal membrane, with which it associates tightly. A comparison with the membrane peroxisomal targeting signal of PMP47 of Candida boidinii revealed a stretch of positively charged amino acids common to both sequences. The role of peroxisomal membrane targeting signals and transmembrane domains in anchoring PMPs to the peroxisomal membrane is discussed.

Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells.

We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides. GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris. GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients. Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light. The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives. This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL is peroxisomal. The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.