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

Saccharomyces cerevisiae Aat2p contains a peroxisomal targeting signal type-1 and localizes to peroxisomes in oleate-grown cells, but not in glucose-grown cells. Here, we have investigated Aat2p from the yeast Hansenula polymorpha, which lacks a recognizable peroxisomal targeting signal. Aat2p tagged with GFP at its C terminus displays a dual cytosol-peroxisome localization in ethanol-grown cells. The partial peroxisomal localization of Aat2p persisted in the absence of the classical cycling receptors Pex5p and Pex7p but Aat2p targeting to peroxisomes was reduced in cells deleted for the matrix protein import factors PEX1, PEX2 and PEX13. Furthermore, we demonstrate that Aat2p targeting to peroxisomes requires Pex20p. Together, our data identify a Pex20p-dependent pathway for targeting Aat2p to peroxisomes.

The N-terminal amphipathic helix of Pex11p self-interacts to induce membrane remodelling during peroxisome fission.

Pex11p plays a crucial role in peroxisome fission. Previously, it was shown that a conserved N-terminal amphipathic helix in Pex11p, termed Pex11-Amph, was necessary for peroxisomal fission in vivo while in vitro studies revealed that this region alone was sufficient to bring about tubulation of liposomes with a lipid consistency resembling the peroxisomal membrane. However, molecular details of how Pex11-Amph remodels the peroxisomal membrane remain unknown. Here we have combined in silico, in vitro and in vivo approaches to gain insights into the molecular mechanisms underlying Pex11-Amph activity. Using molecular dynamics simulations, we observe that Pex11-Amph peptides form linear aggregates on a model membrane. Furthermore, we identify mutations that disrupted this aggregation in silico, which also abolished the peptide's ability to remodel liposomes in vitro, establishing that Pex11p oligomerisation plays a direct role in membrane remodelling. In vivo studies revealed that these mutations resulted in a strong reduction in Pex11 protein levels, indicating that these residues are important for Pex11p function. Taken together, our data demonstrate the power of combining in silico techniques with experimental approaches to investigate the molecular mechanisms underlying Pex11p-dependent membrane remodelling.

Phosphorylation of Pex11p does not regulate peroxisomal fission in the yeast Hansenula polymorpha.

Pex11p plays a crucial role in peroxisomal fission. Studies in Saccharomyces cerevisiae and Pichia pastoris indicated that Pex11p is activated by phosphorylation, which results in enhanced peroxisome proliferation. In S. cerevisiae but not in P. pastoris, Pex11p phosphorylation was shown to regulate the protein's trafficking to peroxisomes. However, phosphorylation of PpPex11p was proposed to influence its interaction with Fis1p, another component of the organellar fission machinery. Here, we have examined the role of Pex11p phosphorylation in the yeast Hansenula polymorpha. Employing mass spectrometry, we demonstrate that HpPex11p is also phosphorylated on a Serine residue present at a similar position to that of ScPex11p and PpPex11p. Furthermore, through the use of mutants designed to mimic both phosphorylated and unphosphorylated forms of HpPex11p, we have investigated the role of this post-translational modification. Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p. Also, no effect on peroxisome inheritance was observed. Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.