1. Volume 4, Issue 1, Pages 21-33 (July 1999)
SGT1 Encodes an Essential Component of the Yeast Kinetochore Assembly Pathway and a Novel Subunit of the SCF Ubiquitin Ligase Complex 
Katsumi Kitagawa, Dorota Skowyra, Stephen J Elledge, J.Wade Harper, Philip Hieter 
Molecular Cell 
Volume 4, Issue 1, Pages (July 1999)
DOI: /S (00)

2. Figure 1
Genetic and Phenotypic Analysis of sgt1 Temperature-Sensitive Mutants
(A) Genetic interactions among SGT1, SKP1, and CTF13. SGT1 overexpression suppresses the temperature sensitivity of the skp1–4 mutant (upper panel). SKP1 or CTF13 overexpression suppresses the temperature sensitivity of the sgt1–3 mutant (lower panel). YPH1161R (skp1–4 cells) containing 2 μm vector alone (pSM217), BPH557 (SKP1 on 2 μm plasmid), BPH556 (CTF13 on 2 μm), BKK17 (SGT1 on 2 μm), CEN vector alone (pRS416), BKK9 (SGT1 on CEN plasmid), or YKK54R (sgt1–3 cells) containing pSM217, BPH557, BPH556, or BPH17 were streaked on SC-uracil plates and incubated at 25°C or 37°C for 4 days.
(B) Fluorescence-activated cell sorting (FACS) profiles of sgt1 mutants. Cells growing in early logarithmic phase at 25°C were split and incubated at either 25°C or 37°C for 6 hr prior to analysis.
(C) Terminal arrest morphologies of sgt1–3 or sgt1–5 cells after 6 hr at 37°C.
Left panel: 4′, 6-diamidino-2-phenylindole (DAPI) staining of DNA and phase contrast photographs of sgt1 cells.
Right panel: Fluorescein isothiocyanate staining of α- and β-tubulin. The fields shown were chosen to emphasize specific phenotypes and not necessarily representative of overall frequencies.
(D) Quantitation of cell and nuclear morphology. Cells were grown to logarithmic phase at 25°C and shifted to 37°C for 6 hr, and nuclear and bud morphology were scored. The numbers shown represent percentages of the total cells scored (300 cells each sample).
(E) Chromosome segregation phenotypes of sgt1 mutants. sgt1 temperature-sensitive alleles were tested for effects on chromosome segregation using a colony color sectoring assay (Koshland and Hieter 1987). Loss of the nonessential marker chromosome gives a red sector in a white colony. sgt1–3 mutants exhibit dramatically increased rates of chromosome loss (highly sectored colonies). In contrast, sgt1–5 mutants exhibit near wild-type rates of chromosome loss.
Molecular Cell 1999 4, 21-33DOI: ( /S (00) )

3. Figure 2 Kinetochore Function of SGT1
(A) FACS profiles of sgt1–3 mad2Δ1 double mutants after temperature shift. Logarithmically growing cells cultured at 25°C were processed or shifted to 37°C for 3 hr before processing.
(B and C) Nondenaturing gels showing CDEIII-binding activity in extracts from sgt1 mutants. Extracts were prepared from congenic wild-type, sgt1–3, or sgt1–5 cells grown continuously at 25°C or shifted to 37°C for 3 hr as indicated. Binding reactions and electrophoresis were performed at 25°C.
(D) Complementation of mutant yeast extracts by addition of recombinant proteins. Extracts (30 μg) from ctf13–30, skp1–4, or sgt1–3 cells were supplemented with buffer alone (lanes 2, 6, and 13) or recombinant proteins as indicated. The amounts of recombinant proteins were normalized by immunoblotting analyses.
Molecular Cell 1999 4, 21-33DOI: ( /S (00) )

4. Figure 3 Association of Sgt1p with Skp1p and SCF Complexes
(A) Coimmunoprecipitation of Sgt1p with Skp1p-3HA from budding yeast extracts. Extracts were prepared from yeast strain YPH1180 expressing Skp1p-3HA from its own promoter (lanes 1 and 2) or from a congenic strain expressing untagged Skp1p (lanes 3 and 4) and immunoprecipitated with HA antibody conjugated sepharose beads. Crude cell lysate and immunoprecipitates were separated on a 10% SDS-PAGE gel, transferred to Immobilon-P (Millipore) membrane, and probed with an anti-Sgt1p antibody.
(B) Coimmunoprecipitation of Skp1p and Cdc53p with 6HA-Sgt1p.
Extracts (10 mg) prepared from cells (YKK330) expressing 6HA-Sgt1p from its own promoter (lane 1) or from a congenic strain expressing untagged Sgt1p (lane 2) were immunoprecipitated with anti-HA antibody. The immunoprecipitates were immunoblotted with anti-Skp1p, anti-Sgt1p, or anti-Cdc53p antibodies.
(C) Comigration of Sgt1p with SCF complexes by gel filtration. Yeast extracts (0.5 mg, F250) were separated by gel filtration on a BioSEC-250 column. Fractions were analyzed for Sgt1p, Skp1p, and Cdc4p by immunoblotting and Cln1-ubiquitin ligase activity using an in vitro assay.
(D) Assembly of SCFCdc4 with Sgt1p in insect cells. The indicated baculoviruses were coinfected into insect cells and Cdc4p immunoprecipitated through a Flag tag. Immune complexes and crude lysates were separated by SDS-PAGE and immunoblotted with the indicated antibodies.
(E and F) Interaction of Sgt1p with Skp1p. The indicated baculoviruses were coinfected into insect cells and either Sgt1p or Skp1p immune complexes purified. Complexes were immunoblotted with the indicated antibodies.
(G) Skp1p binds to sgt1–5 mutant protein, but not sgt1–3 mutant protein. The 35S-methionine-labeled sgt1 mutant proteins were incubated with the Skp1p insect cell extracts at 30°C for 1 hr, and immunoprecipitated with anti-Skp1p antibody.
(H) sgt1–5 cdc53–1 double mutants are inviable at the semipermissive temperature (32°C). The indicated spore clones of a representative tetratype tetrad were grown at 32°C for 3 days.
Molecular Cell 1999 4, 21-33DOI: ( /S (00) )

5. Figure 4 Proteolysis Function of SGT1
(A) Overexpression of Sic1p was lethal in combination with sgt1–5 cells at 33°C. Wild-type, sgt1–5 cells containing a vector only plasmid, or a plasmid expressing Sic1p (pCB24) were grown on galactose plates lacking uracil at 33°C for 4 days.
(B) Wild-type, sgt1–5, sgt1–3, skp1–3, and skp1–4 cells containing pCB24 (GAL-SIC1) were grown in selective SC media containing raffinose and arrested in S phase with 200 mM HU for 3 hr. Galactose (2% w/v) was added for 1.5 hr to induce SIC1 expression, and cells were shifted to 37°C for 30 min. Cells were then resuspended in glucose media at 37°C (2% w/v), and samples were taken at the indicated times, protein prepared, and immunoblotted with anti-Sic1p antibody. The same amount of lysate was used for each lane.
(C and D) sgt1–5 cells are deficient in Cln1p ubiquitin ligase activity in vitro. Wild-type, sgt1–3, and sgt1–5 cells (grown at 25°C) were arrested in S phase with HU prior to shift to the nonpermissive temperature of 37°C. F250 fractions from extracts prepared from these cells were used for in vitro Cln1p ubiquitination assays (see Experimental Procedures) varying either the quantity of lysate ([C], reaction time 60 min) or the reaction time (D). Reaction products were separated by SDS-PAGE, and Cln1-HAp ubiquitin conjugates revealed by immunoblotting with anti-HA antibodies. Blots were reprobed with anti-Skp1p antibodies to demonstrate equal quantities of lysates from each strain.
Molecular Cell 1999 4, 21-33DOI: ( /S (00) )

6. Figure 5 Conservation of Sgt1p in Multicellular Eukaryotes
(A) Multiple Protein Sequence Alignments of Sgt1p homologs. Alignments of five Sgt1p homologs are shown with amino acid identities in black boxes and conservative changes in shaded boxes. Asterisks mark the positions of temperature-sensitive mutations. Sc, S. cerevisiae; Hs, H. sapiens; Dm, D. melanogaster (AA263251); Ce, C. elegans (D1054.3); At, A. thaliana (T44643).
(B) Human Sgt1p can rescue the lethality of an sgt1 null mutant. Yeast strains (YKK39 derivatives) containing either p425MET25 with human SGT1 under MET25 promoter control, p425MET25 alone, or pAD5-SGT1 were streaked onto plates containing 5-FOA, which does not allow growth of cells expressing URA3 (Boeke et al. 1987), and incubated at 30°C for 3 days.
(C) Alignment of TPR motifs within human Sgt1p and yeast Sti1p. Human Sgt1p has three potential TPR motifs at the N-terminal region (R1, 11–44 aa; R2, 45–78 aa; R3, 78–111 aa). This region shows significant homology to the TPR domain of the S. cerevisiae stress-inducible protein Sti1p. The position of the first amino acid residue of each TPR is given to the left. The TPR consensus given at the bottom was reported previously (Chen et al. 1994). φ, amino acids with large hydrophobic side chains.
Molecular Cell 1999 4, 21-33DOI: ( /S (00) )

7. Figure 6 Putative Models for the Linkage of Two Distinct Activities
(A) Skp1p/Sgt1p/Ctf13p recruit an activator protein “X” to the kinetochore.
(B) SCFCtf13 targets ubiquitination, and subsequent degradation, of an inhibitor protein “Y.”
Molecular Cell 1999 4, 21-33DOI: ( /S (00) )