Functionality of Homologous Human Genes in the yeast Saccharomyces cerevisiae Amy Stein Stevens Lab

Eukaryotic cells The V-ATPase is localized to the vacuole/lysosome

H+ ATP  ADP+Pi H+ Ca 2+ H+ H+H+ V-ATPase Acidifies the Vacuole Without a Functioning V-ATPase: –Growth pH 5.0 –No growth on high calcium Vma+ Vma- YEPD CaCl 2 Vacuolar acidfication is essential for cellular ion homostasis

V1V1 V0V0 V 1: hydrolyzes ATP, generates conformational change V 0: the proteolipid ring translocates protons across bilayer, High conservation across organisms The yeast V-ATPase functions by a rotary mechanism

Vo and the Proteolipid Ring Proteolipid Subnits: c : Vma3 c’: Vma11 c’’: Vma Subunit c (Vma3p) Subunit c’ (Vma11p) Subunit c” (Vma16p) Vma3 Vma11 Vma16 Side view Top view

The subunits of the Proteolipid ring are conserved, both between the subunits and between species All other higher Eukaryotes have only 2 species of proteolipid subunits: Vma3p homolog Vma16p homolog Yeast have 3 species of proteolipid subunits: Vma3p Vma11p Vma16p Top view of Proteolipid ring Vma3 Vma11 Vma16 Vma3 Vma16

Will the human proteolipid subunits be able to function in place of the yeast subunits? hVma3 hVma16 hVma3 Vma3 Vma11 Vma16 Will the V-ATPase function in Yeast with: hVma16 in place of Vma16 hVma3 in place of Vma3 hVma3 in place of Vma3 and Vma11 hVma3 and hVma16 in place of Vma3, Vma11 and Vma16

Experimental Design Yeast Plasmid hVma ORF 3’ Flanking Sequence Yeast Promotor Sequence  Yeast vmaΔ Strains  Test for V-ATPase Function 

hVma16 functions in the yeast V-ATPase Vma+ Vma- Recall: YEPD CaCl 2 YEP D CaCl 2 Wild type vmaΔ16 +Vma16 plasmid vmaΔ16 + hVma16 plasmid

Conclusion Vma3 hVma16 Vma11 hVma16 is stably expressed in vmaΔ16 yeast cells hVma16 can assemble with other V-ATPase subunits and for a functional V-ATPase complex

Future Directions Test hVma3 for functionality in vmaΔ3, vmaΔ11 and vmaΔ3Δ11 yeast strains Test hVma3 and hVma16 together in vmaΔ3Δ16 and vmaΔ3Δ11Δ16 yeast strains hVma3 Vma16 hVma3 Vma16 Vma11 hVma3 hVma16 hVma3 hVma16 Vma11

Acknowledgements My Mentor, Dr Laurie Graham Tom Stevens Emily Coonrod Erika Hanson Peter O’Day and everyone who made the SPURs program possible