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.

Ssh1p of Saccharomyces cerevisiae is related in sequence to Sec61p, a general receptor for signal sequences and the major subunit of the channel that guides proteins across the membrane of the endoplasmic reticulum. The split-ubiquitin technique was used to determine whether Ssh1p serves as an additional receptor for signal sequences in vivo. We measured the interactions between the N ub -labeled Ssh1p and C ub -translocation substrates bearing four different signal sequences. The so-determined interaction profile of Ssh1p was compared with the signal sequence interaction profile of the correspondingly modified N ub -Sec61p. The assay reveals interactions of Ssh1p with the signal sequences of Kar2p and invertase, whereas Sec61p additionally interacts with the signal sequences of Mf␣1 and carboxypeptidase Y. The measured physical proximity between Ssh1p and the ␤-subunit of the signal sequence recognition particle receptor confirms our hypothesis that Ssh1p is directly involved in the cotranslational translocation of proteins across the membrane of the endoplasmic reticulum.