Table 1. Synaptotagmin Project Strains Strain Name Genotype Parental Strains 6H11 MATa yorO86c::KanMX met15 LYS2   38c10 MATa yml072c::KanMX met15 LYS2 YAY18 MATalpha yml072c::KanMX MET15 lys2 YAY20 MATalpha ynl087w::KanMX MET15 lys2 YAY30 MATa yml072c::KanMX yorO86c::KanMX met15 LYS2 YAY18 x 6H11 YAY31 MATalpha yml072c::KanMX yorO86c::KanMX MET15 lys2 YAY32 MATa ynl087w::KanMX yor086c::KanMX met15 LYS2 YAY20 x 6H11 YAY33 MATalpha ynl087w::KanMX yor086c::KanMX MET15 lys2 YAY34 MATa ynl087w::KanMX yml072c::KanMX met15 LYS2 YAY20 x 38c10 YAY35 MATalpha ynl087w::KanMX yml072c::KanMX MET15 lys2 YAY36 MATa ynl087w::KanMX yml072c::KanMX yor086c::URA3 met15 LYS2

Synaptotagmins: Why So Many? Sudhof. 2001. Fig. 1.   Structure of the Ca2+-binding sites of the C2A- and C2B-domains of synaptotagmins. The figure shows a generic model for synaptotagmin Ca2+-binding sites that is based on the structure of the C2A- and C2B-domains of Syt 1. The Ca2+-binding sites at the top of the synaptotagmin C2-domains are formed by loops 1 (right blue line) and 3 (left blue line). The C2A-domain ligates three Ca2+ ions via five aspartate and one serine residue, whereas the C2B-domain ligates only two Ca2+ ions. (from Sudhof 2001).

Release of Neurotransmitters and Recycling of Synaptic Vesicles J. Cell. Bio. Sudhof and Jahn. 1991 Figure 2

Nature 375:645 Sudhof. 1995 Figure 3. Synaptic-vesicle and plasma-membrane proteins important for vesicle docking and fusion. Calcium signal displaces synaptotagmin from the SNARE complex thus allowing other proteins and phospholipids to bind and initiate membrane docking and fusion of the two membranes (adapted from Sudhof 1995, Nature 375:645)

Prepared by Jon Schrock Tetrad Dissection of Double Mutants Row of spores Yeast Lab Procedures Prepared by Jon Schrock Oct 2000 Spores were separated by a dissection needle and plated by rows across a selected plate Tetrad Fig. 4a. Tetrad Dissection Fig. 4b. Tetrad Plate

Triple Mutant Construct Figure 5 Homologous Recombination of Triple Mutant Construct PCR Product Transformed a. URA3 yor086c Genome Triple Mutant Construct YNL087w::KAN cassette YML072c::KAN cassette YOR086c::URA cassette Serial Dilution Setup b. 100uL of previous concentration 100uL of previous concentration 100uL of previous concentration Normal Concentration .1 .01 .001

Figure 6

Fig. 7. Double Mutant Check by PCR and Gel Electrophoresis BstE II Marker YAY: 30 31 32 33 34 35 All disruptions were verified by PCR using primers corresponding to sequences outside the disruption sites. (Kanamyacin cassettes replaced the corresponding deletions) PCR reactions were loaded onto gels and ethidium bromide staining of DNA revealed bands for the single and double deletions. (Presence of bands confirmed successful deletion) Checking primers: KAN and YML072c/YOR086c/YNL087w

.1 .001 .1 .001 Double Mutant Strains Wild-type of MAT a and MAT alpha strains YAY30 YAY31 YAY32 YAY33 Single mutants of YNL087w, YML072c, and YOR086c YAY34 YAY35 Double mutants, single mutants, and the wild types were selected and plated onto a YPAD plate at 39 degrees Celsius Double mutants exhibited some growth defects and grew at a slower rate than the single mutants and wild type strains Figure 8

Figure 9. 100mM CaCl2/YPAD 39 Degree .1 .001 Double Mutant Strains .1 .001 Wild-type of MAT a and MAT alpha strains YAY30 YAY31 YAY32 YAY33 Single mutants of YNL087w, YML072c, and YOR086c YAY34 YAY35 Double mutants, single mutants, and the wild types were selected and plated onto a 100mM CaCl2/YPAD Plate at 39 Degrees Celsius Double mutants exhibited some growth defects and also grew at a slower rate as observed with the plate with YPAD only

Cell Fusion Defect by Microscopic Analysis Wild type Fusion defects Veleria et al, 1998 Normal Cell Fusion Cell Fusion Defect Incomplete cell fusion Wild type 80 0 yor086Δynl087Δ 75 9 yor086Δyml072Δ 75 6 ynlo87Δyml072Δ 80 5 Fig. 10a. Cell Fusion Mating Morphology Fig. 10b. Cell Fusion Defects Chart

Fig. 11. Triple Mutant Check BstE II Marker Candidates YAY36 Checking primers: URA3 and KAN

Fig. 12. α Factor Mating Assay YAY36 5 ug 2.5 ug .5 ug 1 ug Fig. 12. α Factor Mating Assay

1.1cm 1.3cm 1.4cm 2.2cm .7cm 1cm 1.2cm 1.5cm 430a YAY36 Table 2. Measurements of α Factor Mating Assay .5ug 1ug 2.5ug 5ug 1.1cm 1.3cm 1.4cm 2.2cm .7cm 1cm 1.2cm 1.5cm 430a YAY36 Average of Experiments (n=10)

430a bar1::HIS3 Replacement YAY36 bar1::HIS3 Replacement Fig. 13. BAR1 Gene Deletion