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3
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Supplementary Material
Supplementary Methods
5 Construction of plasmids:
6 The constructions of plasmids are given below. To construct pSS2

SUM1 , DS13
7 (carrying the original suppressor DNA clone) was digested with Xho I and Pst I (Fig. S2)
8 and the resulting 5.0 kb fragment containing the entire SUM1 ORF, along with its
9 promoter and terminator sequences (1173 bp upstream and 646 bp downstream) was
10 subcloned into Sal IPst I sites of pGAD424, a 2

-based plasmid (Bartel et al ., 1993). To
11 construct YEp24SUM1 and pEGSUM1 , pSS2

SUM1 was digested with Bam HI and
12 Pst I and the 5 kb fragment, containing the entire SUM1 gene (with its own promoter and
13 transcription terminator sequences), was subcloned into Bam HIPst I digested pBlueScript
14 (SK+) (Stratagene Inc.,Sandiego, CA). The resulting plasmid was digested with Bam HI-
15 Sal I, and the 5 kb SUM1 -containing fragment was ligated to Bam HISal I digested YEp24
16 (Botstein et al ., 1979), a 2

and URA3 -based plasmid and Bam HIXho I digested pEG202
17 (Ruden et al.
, 1991), a 2

and HIS3 -based plasmid. YEplac181TUB1 and YEplac181-
18 tub1-1 were constructed as follows. An 1838 bp DNA sequence was amplified by PCR
19 using the primers SS1 (containing 25 bp sequence from a region 196 bp upstream of TUB1
20 ORF and also carrying an engineered Bam HI site; Table S1) and SS2 (containing 19 bp
21 homology to the region 163 bp downstream of TUB1 stop codon and also carrying the
22 engineered Pst I site; Table S1). Genomic DNA from wild-type ( TUB1 ) cells was used as a
23 template for PCR. The resulting PCR product containing the entire TUB1 (including its
24 intron sequence) coding region along with its promoter and terminator region, was
25 digested with Bam HI and Pst I and cloned into the Bam HIPst I sites of YEplac181 to get
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26 YEplac181TUB1 . YEplac181tub1-1 was also created similarly by amplifying the entire
27 tub1-1 ORF along with the promoter region, using genomic DNA from AP27 ( tub1-1 )
28 cells as the template and the same set of primers as used for TUB1 cloning.
29 To construct HST1 in a 2µ-based plasmid, a 1648 bp Eco RIBam HI fragment
30 carrying the entire ORF of HST1 , along with 138 bp sequences upstream of ATG, was
31 amplified by PCR using the following primers. The forward primer SS3 (Table S1) had a
32 21 bp sequence from a region 138 bp upstream of HST1 ORF and also carried an
33 engineered Eco RI site. The reverse primer SS4 (Table S1) had a 21 bp HST1 sequence,
34 beginning from the last codon towards the ORF and also carried an engineered Bam HI
35 site. Genomic DNA of wild-type ( HST1 ) cells was used as template for the amplification.
36 The resulting 1648 bp fragment was digested with Eco RI and Bam HI and cloned into the 2
37 micron plasmid YEplac181 to get YEplac181HST1 . C-terminal tagging of HST1 with six
38 tandem copies of Myc epitope in the centromeric plasmid (YCplac111) as well as in the
39
2µ plasmid (YEplac181) was constructed as follows. The plasmid pUS946 (from Dr. U.
40 Surana) was used that had 6 tandem copies of the ORF for the Myc epitope cloned as a
41 Bam HISal I fragment. The HST1 gene, along with its own promoter, was cloned in frame
42 with the Myc ORF from pUS946 so that the Myc epitope was tagged at the C-terminus of
43 HST1 ORF as follows. A 1.65 kb PCR product was obtained using the genomic DNA of
44 wild-type cells ( HST1 ) as the template and the following primers: forward primer, SS3
45 described above (Table S1) and the reverse primer SS10, having a 21 bp HST1 sequence,
46 beginning from the last codon towards the ORF and an engineered Bam HI site (Table S1).
47 The amplified DNA was digested with Eco RIBam HI and cloned at the Eco RIBam HI site
48 of pUS946 plasmid to obtained pUS946HST1-MYC . The resulting clone was digested
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49 with Eco RIHin dIII and the 1.91 kb fragment carrying the six tandem repeat of Myc
50 epitope at the C-terminus of HST1 was cloned into each of YCplac111 and YEplac181 to
51 obtain YCplac111HST1 MYC and YEplac181HST1 MYC respectively. The expression
52 of Hst1p-Myc was confirmed by western analysis using anti-Myc antibody (Fig. S4b).
53 That the tagged and untagged genes were functional was confirmed from the observation
54 that they complemented the hst1 mutation in the double mutant tub1-1 hst1 to restore its
55 growth at 18

C (data not shown), where the double mutant tub1-1 hst1 does not grow at
56 18

C due to the hst1 mutation (Fig. 3).
57
58 Construction of strains:
59 Deletion/disruptions of SUM1 and HST1 in AP22 and AP27 strains were carried out
60 using appropriate deletion plasmids as follows. HST1 was deleted in both AP22 ( TUB1 )
61 and AP27 ( tub1-1 ) strains with URA3 , for which the deletion plasmid was constructed as
62 follows. The 1648 bp Eco RIBam HI fragment, carrying the entire ORF of HST1 and
63 obtained as described in the supplementary methods, was cloned into pBlueScript (SK+).
64 An internal 693 bp Sph INru I fragment, corresponding to amino acid residues 161-392 of
65 HST1 ORF was replaced with 1.4 kb Sph ISma I fragment containing the URA3 sequence
66 from pUC19U (Sanyal et al ., 1998). The 2.4 kb Eco RISac I fragment carrying the deleted-
67 disrupted HST1 gene was used to create the deletion-disruption of the gene. The deletion
68 was confirmed by PCR analysis, using the primers SS3 and SS4 (Table S1). SUM1 was
69 deleted as follows. A 1074 bp Nru IEco RI internal fragment of SUM1 (corresponding to
70 amino acid residues 554-912) from pSS3 (plasmid having the entire ORF of SUM1
71 contained in the Bam HIXho I fragment cloned at Bam HISal I sites of pBlueScript) was
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72 replaced with 1.5 kb Pvu IIEco RI fragment containing the Kan r sequence from pFA6a-
73 KanMX6 (Wach et al ., 1997). The resulting plasmid was digested with Xba I and Xho I and
74 4258 bp Xba IXho I fragment was used to create the deletion of SUM1 in AP22 and AP27
75 cells. The deletion was confirmed by PCR using the forward and reverse primers SS5 and
76 SS6 (Table S1).
77 C-terminal tagging of SUM1 with thirteen tandem repeats of Myc epitope in the
78 chromosome as well as in the 2µ plasmid (pSS2µSUM1 ) was done according to Longtine
79 et al.
, 1998 as described below. A 2252 bp PCR product was obtained using the plasmid
80 DNA of pFA6a-13Myc-His3MX6 as the template and the following primers: forward
81 primer, SS7 (Table S1; having 42 bp sequence immediate upstream of the stop codon of
82 SUM1 and the next 18 bp sequence from the plasmid) and the reverse primer SS8 (Table
83 S1; having 41 bp sequence immediate downstream of the stop codon of SUM1 and the
84 next 19 bp sequence from the plasmid). The amplified DNA was used to transform AP27
85 ( tub1-1 ) cells or AP27 cells carrying pSS2µSUM1 . The tagging of SUM1 was confirmed
86 by PCR using the forward primer SS9 (Table S1) consisting of 21 bp sequence present
87 119 bp upstream of the SUM1 stop codon and the reverse primer SS6 (Table S1) having
88 23 bp sequence present 113 bp downstream of the SUM1 stop codon. The expression of
89 Sum1p-Myc was confirmed by western analysis using anti-Myc antibody. That the tagged
90 protein was functional was confirmed from the observation that it suppressed the cold-
91 sensitive defect of tub1-1 (data not shown).
92
93
94
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108
109
110
111
112
113
114
103
104
105
106
107
95 DNA sequencing:
96 DNA sequencing was performed by the dideoxy sequencing method using the
97 manufacturer’s instructions. The instrument used was 3130X1 Genetic Analyzer, Applied
98 Biosystems.
99
100
101
102
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115 Supplementary Figure Legends
116 Fig. S1. The immunoblot of Sum1p showing the full length protein (arrow) with its
117 degradation products. Sum1p was detected using anti-Sum1 as described under Materials
118 and Methods. Lanes: 1, extracts of cells expressing single copy SUM1 ; 2, extracts of cells
119 expressing SUM1 from the pSS2µSUM1 plasmid. Similar amounts of total proteins were
120 loaded.
121 Fig. S2. Restriction map of the suppressor plasmid clone (DS13) and its subclones. Each
122 clone was tested for its ability to suppress the growth defect of the double mutant tub1-1
123 mcm22 . Arrows indicate the direction of transcription. Only relevant restriction sites are
124 shown. P
ADH1
refers to the ADH1 promoter. Restriction enzyme abbreviations are as
125 follows: B, Bam HI; Sa, Sau 3AI; H, Hin dIII; P, Pst I; Pv, Pvu II; X, Xho I.
126 Fig. S3 The relative level of Sum1p expressed from pSS2

SUM1 (2μSUM1 ). Wild-type
127 cells (AP22) carrying pGAD424 (2μ) or pSS2 
SUM1 plasmid were grown in SC-Leu
128 medium at 30

C to log phase. Total protein was prepared from the cell extracts by the
129 TCA method. Protein from each cell extract was loaded on SDS-PAGE and
130 immunoblotted with anti-Sum1p antibody. The band intensity of Sum1p was divided by
131 that of Pgk1p, which served as the loading control. The ratio was taken as one for cells
132 containing the empty vector, pGAD424, which is representative of Sum1p levels
133 expressed from the single copy chromosomal gene. The loading was done as follows: 3
 l
134 of each cell extract was loaded for Pgk1 immunoblotting. For Sum1p western, the loading
135 was 20
 l (2

) and 15
 l (pSS2

SUM1 ). The intensity of the Sum1p band in pSS2

-
136 SUM1 lane was accordingly multiplied by the factors 1.33. The band intensities of Sum1p
137 were due to the expression of SUM1 from the chromosomal copy in all the cells as also
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138 due to the expression of SUM1 from plasmid copies in plasmid-bearing cells. These two
139 contributions were taken into account to calculate the relative levels of Sum1p in the
140 fraction of cells that carried 2μ-
SUM1 plasmid.
141 Fig. S4 . High copies of Hst1p do not suppress the benomyl supersensitivity of tub1-1 .
142 AP27 ( tub1-1 ) cells were transformed with YEplac181HST1 (2

HST1 ) and YEplac181
143 (2

) plasmids. Transformant cells were freshly grown on SC-Leu plates and streaked on
144 the same plates containing benomyl at 7.5
 g/ml. Plates were incubated at 23

C for 3 days
145 (no benomyl) and 5 days (+ benomyl). Two independent transformants of YEplac181-
146 HST1 were tested. The tub1-1 transformant carrying pSS2

SUM1 (2

SUM1 ) was used
147 as the positive control. (b) AP22 cells carrying YEplac181 (2µ), YCplac111HST1-MYC
148 (expressing Hst1p-Myc from centromeric plasmid) and YEplac181HST1-MYC
149 (expressing Hst1p-Myc from 2µ plasmid) were grown in SC-Leu medium at 30

C to log
150 phase. Total protein was prepared from the cell extracts by the TCA method. Protein from
151 each cell extract was loaded on SDS-PAGE and immunoblotted with anti-Myc antibody.
152 For the Hst1p westerns, 3 µl of extracts were loaded for each of YEplac181 (2µ) and
153 YCplac111HST1-MYC lanes, while 1.5 µl of protein extract was loaded for the
154 YEplac181HST1-MYC lane. For the Pgk1p western, 3 µl of protein extract was loaded in
155 each case. The band intensities of Hst1p-Myc were divided by those of Pgk1p, which
156 served as the loading control for each extract. The band intensity of the HST1-MYC band
157 in the YEplac181HST1-MYC lane was multiplied by a factor of 2 to normalize with
158 respect to the amount of extract loaded. The ratio was further normalized for plasmid
159 stability (92% for YCplac111HST1-MYC and 93% for YEplac181HST1-MYC ). The ratio
160 of band intensity obtained for cells expressing Hst1p-Myc from centromeric ( CEN )
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161 plasmid was taken as 1 relative to which the increase of Hst1p-Myc levels, expressed from
162 2µ plasmid, was calculated. In two experiments these ratios were 12.5 and 9, the average
163 being 11. In the figure data for the first experiment is shown.
164
165 Fig. S5. Sum1p does not physically associate with

-tubulin .
(A) AP27 ( tub1-1 ) cells
166 carrying pSS2

SUM1 (2

SUM1 ) plasmid were grown in SC-Leu medium at 30

C to
167 log-phase. Sum1p was immunoprecipitated from the cell extracts as described in Materials
168 and Methods. The precipitated materials obtained with (+) or without (-) Sum1 antibody
169 were run on SDS-PAGE and analyzed using anti-Sum1p and anti-

-tubulin antibodies to
170 detect Sum1p and

-tubulin respectively. ‘+’ refers to immunoprecipitates obtained using
171 anti-Sum1p antibody and ‘-’ refers to immunoprecipitates obtained the same way except
172 that no antibody was added. C/E: Total cell extract. For the detection of β-tubulin,
173 polyclonal antibodies against this protein were used in the immunoblot. (B)
174 Untransformed AP22 ( TUB1 SUM1 ) cells, having only the chromosomal copy of the
175 SUM1 gene, were grown to log-phase at 30

C in YEPD medium. Co-immunoprecipitation
176 using cell extracts and anti-Sum1p was performed exactly as described above. Western
177 blots are shown. (C) AP22 and AP22
 sum1 (used as a negative control) were also
178 processed for coimmunoprecipitation studies as described above. Western blots are
179 shown. In both A and B, a band corresponding to

-tubulin was found in the
180 immunoprecipitates (+ antibody lanes) which was, however, also present in the western
181 performed using control SUM1 -deleted (AP22
 sum1) cells in C . This shows that this
182 band was due to a cross-reaction between

-tubulin and anti-Sum1p and does not indicate
183 a specific association between Sum1 and

-tubulin. (D) Further confirmation that Sum1p
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184 and

-tubulin do not coimmunoprecipitate. AP27 ( tub1-1 ) cells carrying pSS2

SUM1
185 (expressing Sum1p from 2

plasmid) or pSS2

SUM1-MYC (expressing Sum1p-Myc
186 from 2

plasmid) were grown to log-phase in SC-Leu medium at 30

C. Similarly, SSY1
187 ( tub1-1 SUM1-MYC ) cells expressing Sum1p-Myc from its chromosomal location and
188 carrying empty vector pGAD424 (2µ) were also grown to log-phase under similar
189 conditions. Sum1p was immunoprecipitated from cell extracts using anti-Myc antibody as
190 described in Materials and Methods. The precipitated materials obtained with (+) or
191 without (-) Myc antibody were run on SDS-PAGE and analyzed using anti-Myc and anti-
192

-tubulin antibodies to detect Sum1p-Myc and

-tubulin respectively. Lane 1-3, tub1-1
193 cells expressing Sum1p-Myc from 2µ-plasmid, Lane 4-6, tub1-1 cells expressing Sum1p-
194 Myc from its chromosomal location, Lane 7-9, tub1-1 cells expressing Sum1p (no Myc
195 tag) from 2µ-plasmid. The band corresponding to IgG heavy chain is also shown in the
196 figure.
197
198 Fig. S6 AP27 ( tub1-1 ) cells were each transformed with YEplac181TUB1 (2

TUB1 ),
199 YEplac181tub1-1 (2

tub1-1 ) and empty plasmid YEplac181 (2μ). Mutant cells carrying
200 these plasmids were grown to log-phase in SC-Leu medium at 30

C. Total protein was
201 prepared from each transformant by TCA method as described in Material and Methods.
202 Immunoblotting was performed using anti-

-tubulin and anti-Pgk1 antibody. For each
203 transformant, the band intensity of α-tubulin subunit was normalized with respect to that
204 of Pgk1p and the value for mutant cells containing the empty vector were defined as 1.0.
205
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206 Fig. S7. tub1-1 and sum1

do not show synthetic growth defects on benomyl plates.
207 Freshly growing cells of AP22, AP22
 sum1, AP27 and AP27
 sum1 were serially diluted
208 and spotted for growth on YEPD plates containing indicated concentrations of benomyl.
209 Plates were incubated at 23

C for two days (no benomyl) and for three days (+benomyl).
10