Figure 1.
Distribution of Predicted Substrate Proteins among the Three Prenyltransferases
Figure 2.
Performance of Different Ranking Schemes for Clusters with Predicted Prenylation Targets
Table 1.
Values of Cluster Medians from Figure 2
Figure 3.
Performance Gain of Ranking Schemes over Standard PrePS without Ranking
Figure 4.
Screenshot of PRENbase Query Interface
Figure 5.
Altered Prenylation under FT Inhibition
Figure 6.
Western Blots and TLC Scanning Results for Rab28 with Radioactive Prenyl Anchor Precursors
Western blot and corresponding scans from TLC linear analyzer of wild-type GST-Rab28-fusion protein translated with [3H]mevalonic acid (lane 1), GST-Rab28 C218A with [3H]mevalonic acid (lane 2), GST-Rab28 with [3H]FPP (lane 3), and GST-Rab28 with [3H]GGPP (lane 4). There is significant incorporation of a product of mevalonic acid as well as FPP, while incorporation of GGPP is not detectable, suggesting that Rab28 is primarily a farnesylation target.
Table 2.
High-Ranked Predicted FTI-Targets (pFs) Sorted by EvOluation Score with Cluster and Taxonomy Statistics
Figure 7.
Western Blots and TLC Scanning Results for FLJ32421 (BROFTI) with Radioactive Prenyl Anchor Precursors
Western blot and corresponding scans from TLC linear analyzer of wild-type GST-FLJ32421-fusion protein translated with [3H]mevalonic acid (lane 1), GST-FLJ32421 C408A with [3H]mevalonic acid (lane 2), GST-FLJ32421 with [3H]FPP (lane 3) and GST-FLJ32421 with [3H]GGPP (lane 4). There is significant incorporation of a product of mevalonic acid as well as FPP, while incorporation of GGPP is close to the detection limit, suggesting that FLJ32421 (BROFTI) is primarily a farnesylation target.
Figure 8.
Western Blots and TLC Scanning Results for a 15 Amino Acid C-terminal Fragment of Prickle1 with Radioactive Prenyl Anchor Precursors
Western blot and corresponding scans from TLC linear analyzer of wild-type GST-ΔPrickle1 fusion protein translated with [3H]mevalonic acid (lane 1), GST-ΔPrickle1 C828A with [3H]mevalonic acid (lane 2), GST-ΔPrickle1 with [3H]FPP (lane 3) and GST-ΔPrickle1 with [3H]GGPP (lane 4). There is significant incorporation of a product of mevalonic acid as well as FPP, while incorporation of GGPP is lower despite a higher total amount of protein in the latter case, suggesting that Prickle1 is primarily a farnesylation target.
Figure 9.
Western Blots and TLC Scanning Results for a 15 Amino Acid C-terminal Fragment of Prickle2 with Radioactive Prenyl Anchor Precursors
Western blot and corresponding scans from TLC linear analyzer of wild-type GST-ΔPrickle2-fusion protein translated with [3H]mevalonic acid (lane 1), GST-ΔPrickle2 C842A with [3H]mevalonic acid (lane 2), GST-ΔPrickle2 with [3H]FPP (lane 3), and GST-ΔPrickle2 with [3H]GGPP (lane 4). There is significant incorporation of a product of mevalonic acid as well as FPP, while incorporation of GGPP is lower despite a higher total amount of protein, suggesting that Prickle2 is primarily a farnesylation target.
Figure 10.
Localization of N-terminal GFP Constructs of Rab28, FLJ32421/BROFTI, Prickle2 (507–844), and RhoA63L in HeLa Cells
HeLa cells were analysed by fluorescence microscopy after transfection with the following constructs: inserts 1, 3, and 4—GFP-Rab28; insert 2—GFP-Rab28 C218A; inserts 5, 7, and 8—GFP-FLJ32421; insert 6—GFP-FLJ32421 C408A; inserts 9, 11, and 12—Prickle2; insert 10—GFP-Prickle2 C841A; inserts 13, 15, and 16—GFP-RhoA63L (as positive control for a geranylgeranylated target); insert 14—GFP-RhoA63L C190S. The GFP-RhoA plasmids were kindly provided by Channing J. Der (University of North Carolina Chapel Hill, Chapel Hill, North Carolina, United States). Nuclei were co-stained with DAPI (blue color).
(A) GFP-Rab28, GFP-FLJ32421, and GFP-Prickle2 are membrane-localized with (4, 8, 12) or without (1, 5, 9) GGTI-298 treatment. Mutation of the Cys in the CaaX box (2, 6, 10) or treatment with FTI-277 (3, 7, 11) cause mislocalization and accumulation of the fusion proteins in the nucleus.
(B) GFP-RhoA is membrane-localized with (15) or without (13) FTI-277 treatment. Mutation of the Cys in the CaaX box (14) or treatment with GGTI-298 (16) cause mislocalization and accumulation of RhoA in the nucleus.
Table 3.
High-Ranked FT Substrates Predicted To Be Unaffected by FT Inhibition