Routing of Hansenula polymorpha alcohol oxidase: an alternative peroxisomal protein-sorting machinery.
Import of Hansenula polymorpha alcohol oxidase (AO) into peroxisomes is dependent on the PTS1 receptor, HpPex5p. The PTS1 of AO (-LARF) is sufficient to direct reporter proteins to peroxisomes. To study AO sorting in more detail, strains producing mutant AO proteins were constructed. AO containing a mutation in the FAD binding fold was mislocalized to the cytosol. This indicates that the PTS1 of AO is not sufficient for import of AO. AO protein in which the PTS1 was destroyed (-LARA) was normally sorted to peroxisomes. Moreover, C-terminal deletions of up to 16 amino acids did not significantly affect AO import, indicating that the PTS1 was not necessary for targeting. Consistent with these observations we found that AO import occurred independent from the C-terminal TPR-domain of HpPex5p, known to bind PTS1 peptides. Synthesis of the N-terminal domain (amino acids 1-272) of HpPex5p in pex5 cells restored AO import, whereas other PTS1 proteins were mislocalized to the cytosol. These data indicate that AO is imported via a novel HpPex5p-dependent protein translocation pathway, which does not require the PTS1 of AO and the C-terminal TPR domains of HpPex5p, but involves FAD binding and the N-terminus of HpPex5p.
Gunkel
Katja
K
Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9750 AA Haren, The Netherlands.
van Dijk
Ralf
R
Veenhuis
Marten
M
van der Klei
Ida J
IJ
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000201:enzymology* / Q000648:ultrastructure; D010843:Pichia / Q000201:enzymology* / Q000648:ultrastructure; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; C000614263:PEX5 protein, Pichia pastoris; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase