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

Pex3p is a peroxisomal membrane protein that is essential for peroxisome bi ogenesis, Here, we show that a conserved stretch of positively charged amin o acids (Arg(11)-X-Lys-Lys-Lys(15)) in the N terminus of Hansenula polymorp ha Pex3p is involved in incorporation of the protein into its target membra ne. Despite the strong conservation, this sequence shows a high degree of r edundancy. Substitution of either Arg(11), Lys(13), Lys(14), Or Lys(15) wit h 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 signifi cant defects in peroxisome assembly and matrix protein import. Additional c hanges in the N terminus of Pex3p, e.g. replacing three or four of the posi tively charged amino acids with negatively charged ones, led to a typical p ex3 phenotype, i.e. accumulation of peroxisomal matrix proteins in the cyto sol and absence of peroxisomal remnants. Also, in these cases, the mutant P ex3p levels mere reduced. Remarkably, mutant Pex3p proteins mere mislocaliz ed to mitochondria or the cytosol, depending on the nature of the mutation, Furthermore, in case of reduced amounts of Pex3p, the levels of other pero xisomal membrane proteins, e.g Pex10p and Pex14p, were also diminished, sug gesting that Pex3p maybe involved in the recruitment or stabilization of th ese proteins tin the membrane).