In Saccharomyces cerevisiae, diploid yeast cells follow a bipolar budding program, which depends on the two transmembrane glycoproteins Bud8p and Bud9p that potentially act as cortical tags to mark the cell poles. Here, we have performed systematic structure-function analyses of Bud8p and Bud9p to identify functional domains. We find that polar transport of Bud8p and Bud9p does not depend on N-terminal sequences but instead on sequences in the median part of the proteins and on the C-terminal parts that contain the transmembrane domains. We show that the guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange factor Bud5p, which is essential for bud site selection and physically interacts with Bud8p, also interacts with Bud9p. Regions of Bud8p and Bud9p predicted to reside in the extracellular space are likely to confer interaction with the N-terminal region of Bud5p, implicating indirect interactions between the cortical tags and the GDP/GTP exchange factor. Finally, we have identified regions of Bud8p and Bud9p that are required for interaction with the cortical tag protein Rax1p. In summary, our study suggests that Bud8p and Bud9p carry distinct domains for delivery of the proteins to the cell poles, for interaction with the general budding machinery and for association with other cortical tag proteins.

BHUM783BUD8prom-myc 6 -BUD8 Δ7-53 fusion in pRS306 this study BHUM784 BUD8prom-myc 6 -BUD8 Δ7-114 fusion in pRS306 this study BHUM785 BUD8prom-myc 6 -BUD8 Δ74-114 fusion in pRS306 this study BHUM786 BUD8prom-myc 6 -BUD8 Δ74-216 fusion in pRS306 this study BHUM787 BUD8prom-myc 6 -BUD8 Δ173-216 fusion in pRS306 this study BHUM788 BUD8prom-myc 6 -BUD8 Δ173-325 fusion in pRS306 this study BHUM789 BUD8prom-myc 6 -BUD8 Δ268-325 fusion in pRS306 this study BHUM790 BUD8prom-myc 6 -BUD8 Δ268-417 fusion in pRS306 this study BHUM791 BUD8prom-myc 6 -BUD8 Δ375-417 fusion in pRS306 this study BHUM792 BUD8prom-myc 6 -BUD8 Δ375-505 fusion in pRS306 this study BHUM793 BUD8prom-myc 6 -BUD8 Δ468-505 fusion in pRS306 this study BHUM794 BUD8prom-myc 6 -BUD8 Δ513-600 fusion in pRS306 this study BHUM795 BUD9prom-myc 3 -BUD9 fusion in pRS426 plasmid coll. H.-U. Mösch BHUM796 BUD9prom-myc 9 -BUD9 fusion in pRS304 this study BHUM797 BUD9prom-myc 9 -BUD9 Δ8-48 fusion in pRS304 this study BHUM798 BUD9prom-myc 9 -BUD9 Δ8-130 fusion in pRS304 this study BHUM799 BUD9prom-myc 9 -BUD9 Δ91-130 fusion in pRS304 this study BHUM800 BUD9prom-myc 9 -BUD9 Δ91-218 fusion in pRS304 this study BHUM801 BUD9prom-myc 9 -BUD9 Δ168-218 fusion in pRS304 this study BHUM802 BUD9prom-myc 9 -BUD9 Δ168-283 fusion in pRS304 this study BHUM803 BUD9prom-myc 9 -BUD9 Δ244-283 fusion in pRS304 this study BHUM804 BUD9prom-myc 9 -BUD9 Δ244-369 fusion in pRS304 this study BHUM805 BUD9prom-myc 9 -BUD9 Δ323-369 fusion in pRS304 this study BHUM806 BUD9prom-myc 9 -BUD9 Δ323-450 fusion in pRS304 this study BHUM807 BUD9prom-myc 9 -BUD9 Δ406-450 fusion in pRS304 this study BHUM808 BUD9prom-myc 9 -BUD9 Δ406-544 fusion in pRS304 this study BHUM809 BUD9prom-myc 9 -BUD9 Δ460-544 fusion in pRS304 this study BHUM810 BUD9prom-myc 9 -BUD9 fusion in pRS305 this study BHUM811 BUD9prom-myc 9 -BUD9 Δ8-48 fusion in pRS305 this study BHUM812 BUD9prom-myc 9 -BUD9 Δ8-130 fusion in pRS305 this study BHUM813 BUD9prom-myc 9 -BUD9 Δ91-130 fusion in pRS305 this study BHUM814 BUD9prom-myc 9 -BUD9 Δ91-130 fusion in pRS305 this study BHUM815 BUD9prom-myc 9 -BUD9 Δ168-218 fusion in pRS305 this study BHUM816 BUD9prom-myc 9 -BUD9 Δ168-283 fusion in pRS305 this study BHUM817 BUD9prom-myc 9 -BUD9 Δ244-283 fusion in pRS305 this study BHUM818 BUD9prom-myc 9 -BUD9 Δ244-369 fusion in pRS305 this study BHUM819 BUD9prom-myc 9 -BUD9 Δ323-369 fusion in pRS305 this study BHUM820 BUD9prom-myc 9 -BUD9 Δ323-450 fusion in pRS305 this study BHUM821 BUD9prom-myc 9 -BUD9 Δ406-450 fusion in pRS305 this study BHUM822 BUD9prom-myc 9 -BUD9 Δ406-544 fusion in pRS305 this study BHUM823 BUD9prom-myc 9 -BUD9 Δ460-544 fusion in pRS305 this study BHUM824 BUD8prom-GFP UV -BUD8 fusion in pRS3426 this study BHUM825 BUD8prom-GFP UV -BUD8 Δ7-53 fusion in pRS3426 this study BHUM826 BUD8prom-GFP UV -BUD8 Δ7-114 fusion in pRS426 this study BHUM827 BUD8prom-GFP UV -BUD8 Δ74-114 fusion in pRS426 this study BHUM828 BUD8prom-GFP UV -BUD8 Δ74-216 fusion in pRS426 this study BHUM829 BUD8prom-GFP UV -BUD8 Δ173-216 fusion in pRS426 this stu...