GGTase3 is a newly identified geranylgeranyltransferase targeting a ubiquitin ligase

Abstract

Protein prenylation is believed to be catalyzed by three heterodimeric enzymes: FTase, GGTase1 and GGTase2. Here we report the identification of a previously unknown human prenyltransferase complex consisting of an orphan prenyltransferase α-subunit, PTAR1, and the catalytic β-subunit of GGTase2, RabGGTB. This enzyme, which we named GGTase3, geranylgeranylates FBXL2 to allow its localization at cell membranes, where this ubiquitin ligase mediates the polyubiquitylation of membrane-anchored proteins. In cells, FBXL2 is specifically recognized by GGTase3 despite having a typical carboxy-terminal CaaX prenylation motif that is predicted to be recognized by GGTase1. Our crystal structure analysis of the full-length GGTase3–FBXL2–SKP1 complex reveals an extensive multivalent interface specifically formed between the leucine-rich repeat domain of FBXL2 and PTAR1, which unmasks the structural basis of the substrate-enzyme specificity. By uncovering a missing prenyltransferase and its unique mode of substrate recognition, our findings call for a revision of the ‘prenylation code’.

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Fig. 1: PTAR1, an orphan prenyltransferase α-subunit, binds FBXL2 and RabGGTB.
Fig. 2: GGTase3 geranylgeranylates FBXL2 and is required for its localization to cellular membranes.
Fig. 3: Overall structure of GGTase3–FBXL2–SKP1 complex.
Fig. 4: Structural features of GGTase3 and FBXL2.
Fig. 5: Multivalent FBXL2–GGTase3 interface.

Data availability

Structural coordinates and structural factors have been deposited to the Protein Data Bank (PDB) under accession number 6O60. Source data for Fig. 2a,b and Supplementary Fig. 2b are available with the paper online. All other data are available from the authors upon request.

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Acknowledgements

The authors thank G. Rona for his contribution, C. Fierke, J. Ramalho and M. Seabra for reagents, T.R. Hinds for Octet BLI analysis and M. Bergo for critically reading the manuscript. M.P. is grateful to T.M. Thor for continuous support. This work was funded by grants from the National Institutes of Health (NIH) (nos. R01-GM057587 and R01-CA076584) to M.P. and a fellowship from the T32-CA009161 (Levy) grant to A.M. M.P. and N.Z. are investigators with the Howard Hughes Medical Institute.

Author information

S.K. and M.P. conceived the project. S.K. designed and performed most biochemical, molecular biology and cell biology experiments. H.W. and N.Z. conceived and performed most protein purifications and all crystallization experiments. A.M., K.J. and H.H. performed some of the biochemical experiments. N.F. and M.R.P. helped with the initial microscopy experiments. M.P. and N.Z. directed and coordinated the study and oversaw the results with S.K. and H.W. All authors discussed the results and commented on the manuscript.

Correspondence to Ning Zheng or Michele Pagano.

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Competing interests

M.P. is a consultant for BeyondSpring Pharmaceuticals and a member of the scientific advisory boards of CullGen, Inc. and Kymera Therapeutics. N.Z. is a member of the scientific advisory board of Kymera Therapeutics. The authors declare no other competing interests.

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Peer review information: Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 PTAR1 specifically binds FBXL2 and RabGGTB.

(a) IMR90 diploid fibroblasts and HEK293T cells were transfected with the indicated siRNA oligos. Seventy-two hours after siRNA transfection, cells were harvested for immunoblotting as indicated. NS: non-silencing. (b) HEK-293T cells were co-transfecetd with FLAG-tagged PTAR1 and either GFP-tagged FBXL15, GFP-tagged H-Ras, or GFP-tagged FBXL2. Twenty-four hours post-transfection, cells were harvested for immunoprecipitations and immunoblotting as indicated. (c) HEK-293T cells were transfected with the indicated siRNA oligos for 72 hours before cells were harvested for immunoblotting as indicated. NS: non-silencing, s.e.: short exposure, l.e: long exposure. (d,e) HEK-293T cells were transfected with FLAG-tagged PTAR1 and the indicated GFP-tagged substrates of FTase or GGTases. Twenty-four hours post-transfection, cells were harvested for immunoprecipitations and immunoblotting as indicated. The asterisks indicate bands visualized only after long exposures. s.e.: short exposure, l.e: long exposure. (f) HEK-293T cells were transfected with the indicated siRNA oligos for 48 hours followed by transfection with indicated cDNAs. Sixteen hours after transfection of the cDNAs, cells were lysed, and immunoprecipitations were performed with an anti-GFP antibody followed by immunoblotting as indicated. WCE: Whole cell extract. All experiments were repeated at least three times.

Supplementary Figure 2 In vitro geranylgeranylation of FBXL2 by GGTase1 and delocalization of FBXL2(C420S).

(a) Decreasing amounts of BSA (600, 300, 150, 75, or 37.5 ng) and 100 ng of either purified recombinant untagged (UT) GGTase3, or purified recombinant tagged (T; MBP-PTAR1 and GST-RabGTTB) GGTase3, were subjected to SDS-PAGE and stained with Coomassie Blue. (b) Top panel: The indicated amounts of purified FBXL2 and CDC42 were incubated with 100 ng of purified recombinant GGtase1 to carry out in vitro geranylgeranylation assay using tritiated [H3]-GGPP as described in methods. CDC42, a known GGTase1 substrate, was used as a positive control. Geranylgenranylation of FBXL2 was measured by determining the transfer of [3H]-GGPP onto purified FBXL2 and CDC42 by GGTase1 and plotted as µM/min using CPM counts. Each data point represents the GGTase1 activity (mean+/− SD)/µM/min of three technical replicates. Bottom panel: Bar graphs show in vitro geranylgeranylation assay carried out and measured as in Fig. 2b, using 10 µM of purified FBXL2, FBXW7, or CDC42 and 100 ng of purified GGTase1. Control no enzyme samples were run as biological duplicates and GGtase1 samples were run as biological triplicates. Error bar shows SEM. (c) Hela cells were transiently transfected with either GFP-tagged FBXL2 or GFP-tagged FBXL2(C420S) cDNAs. After sixteen hours, live cell imaging was carried with a LSM510 confocal microscope using a 63X objective. Images show representative frames of three independent experiments. Bar size: 10 μm.

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Supplementary Figs. 1 and 2, Supplementary Notes 1 and 2, Supplementary Data Set 1

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