Table 3. Prevalence of Ste5 in punctate foci.

Strain/Gene/Protein1	%Punctate spots2	%Nuc. Accum3	%Cortical4
A. ADH1prom-SSA1-GFP
EY957 bar1D RT (N = 2)	0 +/- 0	0.8 +/- 0.4	0 +/- 0
EY3141 ssa1D ssa2D RT (N = 3)	0 +/- 0	82.7 +/- 5.5	0 +/- 0
B. GFP database of Huh et al [98].5
yeastgfp.yeastgenome.org/display
LocImage.php?loc = 15528):
S288c + Ssa1-GFP (32 cells)	3 (1 cell)	0	0
yeastgfp.yeastgenome.org/display
ocImage.php? loc = 13306:
S288c + Ssa2-GFP (71 cells)	5.6 (4 cells)	1.4 (1 cell)	0
C. ste5D + STE5promSTE5-MYC9
(indirect immunofluorescence)
W303a ste5D, 30°C	0 +/- 0	23 +/- 5.3	0.3 +/- 0.3
p-value, N = 4	1
W303a ste5D, a factor, 30°C	0.2+/-0.2	8.9+/-3.3	20.3+/-3.4
p-value, N = 7	0.424114
W203a ste5D, a factor, RT	0	0	29.4
no p-value, N = 1
D. STE5 + CUP1prom-STE5-MYC9
(indirect immunofluorescence)
W303a,S288c, 30°C	0 +/- 0	7.7 +/- 7.7	0 +/- 0
p-value, N = 2	1
E. STE5 + STE5-MYC9-2m
(indirect immunofluorescence)
W303a, 30°C	0 +/- 0	19.5 +/- 7.6	0.7 +/- 0.7
p-value, N = 4	1
F. ste5D + Ste5(1–242)-GFP-GFP
RING-H2 (live)6
Ste5(1–242)-GFP2, RT	11.4 +/- 3.96	84.1 +/ -6.7	37.5 +/- 19.2
p-value, N = 77	0.040835
Ste5(1–242)-GFP2, 30°C	5.3 +/- 3.4	80.1 +/- 4.8	4.3 +/- 4.3
p-value, N = 8	0.30671
Ste5(1–242)-GFP2, 37°C	16.3 +/- 5.2	85.7+/-5.4	0 +/- 0
p-value, N = 2	0.001903
G. ste5D + ADH2prom-TAgNLS-
-GFP-GFP (live)
W303a ste5D, 30°C	0 +/- 0	100 +/- 0	0 +/- 0
p-value, N = 3	1
H. ste5D + ADH2prom-TAgNLS-
NES(PKI)-GFP-GFP (live)
W303a ste5D 30°C	0 +/- 0	0 +/- 0	0 +/- 0
p-value, N = 2	1
I. ste5D + STE5prom- STE5-GST
(indirect immunofluorescence)
Ste5-GST, RT	1.4 +/- 1	0.9 +/- 0.9	32.3+/-11.6
p-value, N = 5	0.236212
Ste5-GST, RT, a factor	1.8 +/- 1.8	19.0+/-19.0	28.6+/-4.8
p-value, N = 3	0.219944
Ste5-GST, 37oC (N = 1)	0	5	5
Ste5-GST, 37°C, a factor (N = 1)	0	5	20
J. ste5D + STE5prom-TAgNLS-
-STE5-GST
W303a ste5D, 30°C	0 +/- 0	98.3 +/- 1.7	0 +/- 0
p-value, N = 2	1
K. ste5D + STE5prom-ste5-L610/614/
634/637A-MYC9-2m
W303a ste5D, 30°C	1.4 +/- 1.4	0 +/- 0	0 +/- 0
p-value, N = 3	0.014993
L. ste5D + STE5prom-TAgNLS-ste5-
-L610/614/634/637A-MYC9-CEN
W303a ste5D, 30°C	2 +/- 2	74	0
p-value, N = 2	0.078141
M. ste5D + STE5prom-ste5-
-L610/614/634/637A-GST-2m
W303a ste5D 30°C	8.7	78.9	0
W303a ste5D, 30°C, + aF	10	87	0
no p-value, N = 1
N. ste5D + STE5prom-ste5-
-L610/614/634/637A-MYC9-2m
W303a ste5D, 30°C	6.9+/-3.8	52.0+/-27	0 +/- 0
p-value, N = 3	0.048718
O. ste5D + STE5prom-Ste5-
-L610/614/634-/637A-Myc9-2m
+ GAL1prom-SSA1-GFP
W303a ste5D, 30°C	43.2+/- 4.6	9.6+/-6.3	0 +/- 0
p-value, N = 4	0.000082
P. ste5D + STE5prom-Ste5-
L582/585A-MYC9-2m
+ GAL1prom-SSA1-GFP
W303a ste5D 30°C	0	24.2	0
no p-value, N = 1

¹Cells were grown in SC selective medium containing 2% dextrose, either lacking uracil, tryptophan and /or leucine at 30°C or at the indicated temperature. Cells were grown to logarithmic phase at an A₆₀₀ of 0.2–0.5 adjusted for equal cell density, ~A₆₀₀ 0.5 then treated with a factor. Where indicated, cells were transferred to pre-warmed medium at 37°C and incubated for 3 hours. For the GAL1 promoter, cells were pre-grown in pre-warmed medium containing 2% raffinose prior to being transferred to pre-warmed medium containing 2% galactose and then grown for 5 hours.

²Cells were fixed with 10% formaldehyde and processed for indirect immunofluorescence to detect Myc tagged proteins and DAPI stained DNA or were visualized live for GFP tagged proteins as described (S3, S4).

The analysis used captured images from published and unpublished work.

²The % punctate spots is the percentage of cells that harbored 1 or more spots.

³Nuclear accumulation means that the 9E10 signal in the nucleus is equal to or greater than the 9E10 signal in the cytoplasm. In some instances, nuclear localization is an estimation done without DAPI co-staining or localization of a protein known to be nuclear. Nuclear exclusion means that the 9E10 signal is less than the 9E10 signal in the cytoplasm.

⁴The cortical accumulation in all cases was asymmetric and if a shmoo was present, it was located at the shmoo tip.

⁵The Yeast GFP fusion database of Erin O’Shea and Jonathan Weissman laboratories [98] is hosted by SGD at yeastgfp.yeastgenome.org. The localization of Ssa1-GFP and Ssa2-GFP appears to have an uneven lacy pattern without obvious nuclear accumulation. Note that nuclear accumulation is an estimate because there is no DAPI.

⁶Ste5(1–242)-GFP2 was visualized live in cells grown at RT, 37°C and by indirect immune-fluorescence on fixed cells that were grown at 30°C. The GFP⁺ cytoplasmic pool is more prominent than the nuclear pool in fixed cells than in live cells.

⁷Values shown are the mean +/- standard error.

⁸p-values were calculated with a student’s T-test (two independent means, two-tailed). The p-values were calculated by comparing the punctate foci values of the various Ste5 derivatives with those of ste5D + STE5-MYC9-2m grown at 30°C (i.e. 0,0,0,0,0 (N = 5)).