In Saccharomyces cerevisiae four transporters, Tpo1p-Tpo4p, all members of the major facilitator superfamily, have been shown to confer resistance to polyamines. It was suggested that they act by pumping their respective substrate into the lumen of the vacuole depending on the proton gradient generated by the VATPase. Using sucrose gradient ultracentrifugation we found that an hemagglutinin (HA)-tagged Tpo1p as well as its HA-tagged Tpo2p-4p homologues co-localize with plasma membrane markers. Because the HA-tagged Tpo1p carrier protein proved to be functional in conferring resistance to polyamines in TPO1 knockouts, a function of Tpo1p in transport of polyamines across the plasma membrane seemed to be likely. The polyamine transport activity of wild type cells was compared with the respective activity of a TPO1 knockout strain. The results obtained strongly suggest that Tpo1p is a plasma membrane-bound exporter, involved in the detoxification of excess spermidine in yeast. When studying polyamine transport of wild type cells, we furthermore found that S. cerevisiae is excreting putrescine during the fermentative growth phase.

A database of 5448 peptide tandem mass spectra acquired in a quadrupole time-of-flight mass spectrometer was generated for peptides derived from proteins digested with trypsin. Peptides were identified from their mass spectra by the Mascot algorithm. Statistical models were then used to investigate factors influencing the abundance of ions formed. Separate models were formulated for b and y ions as it was thought that different factors may influence the formation of each type of ion. Several factors were found to have a highly significant influence on the abundance of ions formed. These include the actual mass of the ion formed after fragmentation as well as the location of the cleavage. The composition of the fragmenting peptide was also found to be important, and amino acids either side of the fragmentation site influenced the abundance of ions produced. To increase understanding of fragmentation mechanisms, the effect of several physicochemical properties of these residues was also investigated in a separate model. In conclusion, the models formulated for b and y ions provide useful characterization of the abundance of ions formed, and this information could be used to develop improved algorithms for peptide identification.