Figure EV1. GGTase‐III has no prenyltransferase activity on known prenyltransferase substrates.

• A–D
  Prenylation activity of GGTase‐III on Ras, Rho, and Rab proteins. (A) His₆‐H‐Ras (5 μM) was incubated with FTase (50 nM) or GGTase‐III (50 nM) and ³H‐FPP (1 μM) at 37°C. Reactions were stopped at the indicated time points, and the amount of ³H‐farnesyl transferred to H‐Ras was quantified by scintillation counting (mean ± SEM, n = 3). (B) His₆‐RhoA (5 μM) was incubated with GGTase‐I (50 nM) or GGTase‐III (50 nM) and ³H‐GGPP (1 μM), and the amount of ³H‐geranylgeranyl transferred to RhoA was quantified (mean ± SEM, n = 3). (C) His₆‐Rab1A (5 μM) was incubated with RabGGTase (50 nM) or GGTase‐III (50 nM) and ³H‐GGPP (1 μM) in the absence or presence of either His₆‐REP1 (100 nM) or His₆‐REP2 (100 nM), and the amount of ³H‐geranylgeranyl transferred to Rab1A was quantified (mean ± SEM, n = 3). (D) His₆‐Rab5A and His₆‐Rab11A were incubated with RabGGTase (50 nM) or GGTase‐III (50 nM) and ³H‐GGPP (1 μM) in the presence of His₆‐REP1 (100 nM), and the amount of ³H‐geranylgeranyl transferred to Rab5A and Rab11A was quantified (mean ± SEM, n = 3).
• E
  REP pull‐down assay. Recombinant His₆‐REP1 or His₆‐REP2 was incubated with glutathione Sepharose beads coated with GST, GST‐RabGGTase, or GST‐GGTase‐III. Bound His₆‐REP proteins were analyzed by immunoblotting with anti‐His₆ antibody.

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