Biochemical analysis of γ-TuRC components.

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Biochemical analysis of γ-TuRC components. Biochemical analysis of γ-TuRC components. (A) Molecular mass distribution showing a general schematic of fractionation profiles based on western analysis. This trace is absorbance units versus fraction number obtained by Fast Protein Liquid Chromatography (FPLC). High molecular mass γ-TuRC peaks occur in fractions 17 and 18. Molecular size markers at 2000 kDa and 669 kDa are shown. (B) Western profiles of whole-cell extracts fractionated by FPLC. Whole-cell extracts were prepared from cells expressing integrated Alp4-HA (strain LV15), Alp6-HA (strain LV16), GCP3-HA-His (JP32-2) or GCP2-HA (JPZO46) and equalized to ∼30 mg/ml, collected in 0.5 ml fractions from a Superose 6 Column. The γ-tubulin profile shown is from the Alp4-HA strain, done in parallel. GCP3-HA-His was probed with anti-HA. (C) Western of whole cell extracts fractionated by FPLC. Profiles of cells containing integrated prep90×FLAGHis/GCP2 (strain JPZO44), as analyzed in Fig. 3. Tagged constructs were probed with anti-FLAG and the γ-tubulin profile is from the prep90×FLAGHis/GCP2 strain, done in parallel. GCP2 high molecular mass fractions are shifted right, indicating limited assembly into the high molecular mass γ-TuRC. Rather, GCP2 fractionates primarily as smaller complexes. The FLAG-His tag does not disrupt Alp4 function in our growth assays (not shown). Timothy D. Riehlman et al. J Cell Sci 2013;126:4406-4413 © 2013. Published by The Company of Biologists Ltd