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. Author manuscript; available in PMC: 2015 Feb 10.
Published in final edited form as: Dev Cell. 2014 Jan 23;28(3):268–281. doi: 10.1016/j.devcel.2013.12.013

Figure 4. The conserved GLEBS motif in BuGZ binds and stabilizes Bub3 and promotes Bub3 kinetochore loading and function.

Figure 4

A. Sequence alignment of human BuGZ amino acids 335–372 with the known GLEBS motifs from the indicated proteins. The completely conserved amino acids, EE, are boxed.

B. Flag-BuGZ, but not Flag-luciferase (Flag-Lucifer) or Flag-BuGZAA (with the conserved EE boxed in A mutated to AA), pulled down endogenous Bub3 in HeLa cells. Loading control, GAPDH.

C. Expression of the RNAi-insensitive versions of RFP-BuGZ, but not RFP or RFP-BuGZAA, stabilized both endogenous Bub3 and stably expressed GFP-Bub3 in HeLa cells treated with control or BuGZ siRNA. Note that the levels of rescue RFP-BuGZ and RFP-BuGZAA exceeded those of the endogenous BuGZ by ~2-fold, but only RFP-BuGZ caused a modest increase of Bub3.

D. Expression of the RNAi-insensitive RFP-BuGZ, but not RFP-BuGZAA or RFP, rescued the kinetochore localization of Bub3 in HeLa cells treated with BuGZ siRNA. The appreciably stronger kinetochore Bub3 signal in cells treated with control or BuGZ siRNA and expressing RFP-BuGZ is consistent with the increased Bub3 protein levels in these cells compared to other controls (see C). CREST was used to mark centromeres. The boxed kinetochore was enlarged to the bottom right of each merged images. Scale bar, 5 μm.

E. Expression of RNAi-insensitive Flag-BuGZ, but not Flag-BuGZAA or Flag-luciferase, stabilized Bub3 upon BuGZ RNAi. Note the levels of rescue Flag-BuGZ and Flag-BuGZAA exceeded that of endogenous BuGZ by ~2-fold, but only Flag-BuGZ caused a modest increase of Bub3. The color code for each lane corresponds to the bars in the graph in (F).

F. Stabilization of Bub3 by Flag-BuGZ significantly reduced the mitotic index in HeLa cells treated with BuGZ siRNA. The elevated Bub3 levels in cells expressing the rescue Flag-BuGZ (see E) correspond to the moderate increase in the mitotic index (compare yellow and dark blue bars to the red bar). ~300 cells were counted for each experiment and condition.

G. Flag-BuGZ, but not Flag-BuGZAA, rescued the chromosome misalignment in cells treated with BuGZ siRNA. Flag-luciferase served as a negative control. ~40 mitotic cells were counted for each experiment and condition.

Error bars, SEM. Student’s t-test: ***p<0.001 from triplicates.