Cells acquire free metals through plasma membrane transporters. But, in natural settings, sequestering agents often render metals inaccessible to transporters, limiting metal bioavailability. Here we identify a pathway for metal acquisition, allowing cells to cope with this situation. Under limited bioavailability of Mg2+, yeast cells upregulate fluid-phase endocytosis and transfer solutes from the environment into their vacuole, an acidocalcisome-like compartment loaded with highly concentrated polyphosphate. We propose that this anionic inorganic polymer, which is an avid chelator of Mg2+, serves as an immobilized cation filter that accumulates Mg2+ inside these organelles. It thus allows the vacuolar exporter Mnr2 to efficiently transfer Mg2+ into the cytosol. Leishmania parasites also employ acidocalcisomal polyphosphate to multiply in their Mg2+-limited habitat, the phagolysosomes of inflammatory macrophages. This suggests that the pathway for metal uptake via endocytosis, acidocalcisomal polyphosphates and export into the cytosol, which we term EAPEC, is conserved.

We have studied Hansenula polymorpha Pex5p and Pex20p, peroxins involved in peroxisomal matrix protein import.

Pyruvate carboxylase (Pyc1p) has multiple functions in methylotrophic yeast species. Besides its function as an enzyme, Pyc1p is required for assembly of peroxisomal alcohol oxidase (AO). Hence, Pyc1p-deficient cells share aspartate auxotrophy (Asp-) with a defect in growth on methanol as sole carbon source (Mut-). To identify regions in Hansenula polymorpha Pyc1p that are required for the function of HpPyc1p in AO assembly, a series of random mutations was generated in the HpPYC1 gene by transposon mutagenesis. Upon introduction of 18 mutant genes into the H. polymorpha PYC1 deletion strain (pyc1), four different phenotypes were obtained, namely Asp- Mut-, Asp- Mut+, Asp+ Mut-, and Asp+ Mut+. One mutant showed an Asp+ Mut- phenotype. This mutant produced HpPyc1p containing a pentapeptide insertion in the region that links the conserved N-terminal biotin carboxylation domain (BC) with the central transcarboxylation (TC) domain. Three mutants that were Asp- Mut- contained insertions in the TC domain, suggesting that this domain is important for both functions of Pyc1p. Analysis of a series of constructed C-terminal and N-terminal truncated versions of HpPyc1p showed that the TC domain of Pyc1p, including the region linking this domain to the BC domain, is essential for AO assembly.

d-Amino acid oxidase (DAO) is an important flavo-enzyme that catalyzes the oxidative deamination of d-amino acids into the corresponding alpha-keto acid, ammonia and H(2)O(2). We identified two amino acid oxidases in the methylotrophic yeast Pichia pastoris: Dao1p, which preferentially uses d-alanine as a substrate, and Dao2p, which uses d-aspartate as a preferred substrate. Dao1p has a molecular mass of 38.2 kDa and a pH optimum of 9.6. This enzyme was localized to peroxisomes, albeit a typical peroxisomal targeting signal is lacking. Interestingly, P. pastoris mutant strains, defective in the enzyme pyruvate carboxylase, showed a pronounced growth defect on d-alanine, concomitant with a significant reduction in Dao1p activity relative to the wild-type control. This indicates that pyruvate carboxylase functions in import and/or activation of P. pastoris Dao1p.