Routing ofHansenula polymorphaAlcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery

Abstract

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.

Hansenula polymorpha and Saccharomyces cerevisiae Pex5p's recognize different, independent peroxisomal targeting signals in alcohol oxidase

“…Hansenula polymorpha NCYC 495 (leu2) [7], S. cerevisiae CEN.PK2-1D (MATa, leu2 [3,112]; ura3-52; his3-D1; MAL2-8 C ; SUC2) [8] and CEN.PK700 (MATa, leu2 [3,11]; ura3-52his3-D1; MAL2-8 C ; SUC2; pyc1 [41,3501]::loxP-Kan-loxP; pyc2 [41,3496]::loxP-Kan-loxP), in which PYC1 and PYC2 were deleted, were used. All S. cerevisiae strains were derived from these strains, except for UTL7A pex5 [9] (see Table 1).…”

“…Plasmid YEplac195-P TPI1 AOX-LARA-T CYC1 , encoding AO in which the C-terminal phenylalanine was replaced by alanine (AO mut ) was made by replacement of the PstI (T4 DNA polymerase-blunt ended)/SalI AOX-fragment in YEplac195-P TPI1 AOX-T CYC1 with a 1105-bp SalI/StuI fragment of pHIPX1-AOX-LARA [3].…”

“…It contains a PTS1 ( -ARF) that is sufficient to direct a reporter protein to peroxisomes [1,2], but is redundant for AO sorting. Nevertheless, AO import depends on Pex5p, but its C-terminal TPR domain is not required [3]. Surprisingly, the cytosolic enzyme pyruvate carboxylase (Pyc1p) is essential for AO sorting and activation, most likely by being involved in FAD-binding to AO monomers [4].…”

Hansenula polymorpha Aat2p is targeted to peroxisomes via a novel Pex20p‐dependent pathway

“…Pex7p functions in cargo binding, whereas the coreceptors are required for membrane docking and recycling. Evidence exists for a third pathway that is independent of a PTS1, but requires the N‐terminal domain of Pex5p 16, 17. Sometimes referred to as the PTS3 pathway, to date no PTS3 consensus sequence has been described 18.…”

Octameric alcohol oxidase dissociates into stable, soluble monomers upon incubation with dimethylsulfoxide

“…The AO biosynthetic pathway-from the synthesis of inactive AO monomers in the cytosol to the formation of enzymatically active AO octamers in the peroxisomal matrix-has been the topic of extensive research, which revealed that FAD binding is not only important for the enzyme activity of AO, but also essential to allow import of the protein into peroxisomes [2][3][4]. The association of FAD to newly synthesized AO monomers requires the cytosolic protein pyruvate carboxylase (HpPyc1p) [5].…”