stretch of positively charged amino acids at the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into the peroxisomal membrane

• Published in: The Journal of biological chemistry 2000-04, Vol.275 (14), p.9986-9995
• Main Authors: Baerends, R.J.S, Faber, K.N, Kram, A.M, Kiel, J.A.K.W, Klei, I.J. van der, Veenhuis, M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Amino Acid Substitution; Animals; ATP-Binding Cassette Transporters; cell membranes; Consensus Sequence; Conserved Sequence; cytochemistry; cytosol; DNA Primers; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Green Fluorescent Proteins; Hansenula; Hansenula polymorpha; immunocytochemistry; Intracellular Membranes - metabolism; Intracellular Membranes - ultrastructure; Luminescent Proteins - analysis; Luminescent Proteins - genetics; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; mitochondria; Molecular Sequence Data; Mutagenesis, Site-Directed; mutants; Peroxins; peroxisomes; Peroxisomes - metabolism; Peroxisomes - ultrastructure; Pex3 protein; Pex3p protein; Pichia - genetics; Pichia - growth & development; Pichia - metabolism; Plasmids; proteins; Recombinant Fusion Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Deletion; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.14.9986

Pex3p is a peroxisomal membrane protein that is essential for peroxisome biogenesis. Here, we show that a conserved stretch of positively charged amino acids (Arg(11)-X-Lys-Lys-Lys(15)) in the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into its target membrane. Despite the strong conservation, this sequence shows a high degree of redundancy. Substitution of either Arg(11), Lys(13), Lys(14), or Lys(15) with uncharged or negatively charged amino acids did not interfere with Pex3p location and function. However, a mutant Pex3p, carrying negatively charged amino acids at position 13 and 15 (K13E/K15E), caused moderate but significant defects in peroxisome assembly and matrix protein import. Additional changes in the N terminus of Pex3p, e.g. replacing three or four of the positively charged amino acids with negatively charged ones, led to a typical pex3 phenotype, i.e. accumulation of peroxisomal matrix proteins in the cytosol and absence of peroxisomal remnants. Also, in these cases, the mutant Pex3p levels were reduced. Remarkably, mutant Pex3p proteins were mislocalized to mitochondria or the cytosol, depending on the nature of the mutation. Furthermore, in case of reduced amounts of Pex3p, the levels of other peroxisomal membrane proteins, e.g. Pex10p and Pex14p, were also diminished, suggesting that Pex3p maybe involved in the recruitment or stabilization of these proteins (in the membrane).