MUTATIONAL ANALYSIS OF THE N-TERMINAL TOPOGENIC SIGNAL OF WATERMELON GLYOXYSOMAL MALATE-DEHYDROGENASE USING THE HETEROLOGOUS HOST HANSENULA-POLYMORPHA

We have studied the significance of the N-terminal presequence of wate rmelon (Citrullus vulgaris) glyoxysomal malate dehydrogenase [gMDH; (S )-malate:NAD+ oxidoreductase; EC 1.1.1.37] in microbody targeting. The yeast Hansenula polymorpha was used as heterologous host for the in v ivo expression of various genetically altered watermelon MDH genes, wh ose protein products were localized by immunocytochemical techniques. It is shown that the presequence of gMDH is essential and sufficient f or peroxisomal targeting; it can target the mature part of the mitocho ndrial MDH to microbodies, whereas deletion of the presequence results in accumulation of the mature form of gMDH in the cytosol. Alignment of the N termini of several peroxisomal proteins that are assumed to c ontain a peroxisomal targeting signal at the N terminus (PTS2) suggest ed the consensus sequence RL-X5-HL. A similar motif is present in the presequence of watermelon gMDH-namely, 10RI-X5-17HL. Mutational analys is revealed that substitutions of 10RI into DD or 17HL into DE destroy ed the topogenic information, whereas substitutions of 25M into I and 26EE into LV did not. By combining our data with recent analyses of ot hers on the presequences of mammalian thiolases, it is concluded that the peroxisomal targeting information of PTS2 is contained in the cons ensus sequence RL/I-X5-HL. In contrast to the higher plant and mammals , the Hansenula yeast peroxisomes seem to lack an enzyme capable of re moving microbody presequences of higher eukaryotes.