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. 2018 Jan 3;9:44. doi: 10.1038/s41467-017-02110-1

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — GtgE specifically cleaves the inactive form of Rab32-family members. a Substrate range of GtgE is restricted to Rab32 family members. Proteolytic gel shift assay of the Rab32-subfamily and Rab23. b Nucleotide-dependent GtgE-mediated (G, 8 nM) cleavage of Rab32_FL (R, 8 µM) bound to GDP, GTP or GppNHp in a time-dependent SDS-PAGE gel shift assay. c Direct comparison of the intrinsic GTP-hydrolysis of Rab32:GTP (black) and the GtgE-mediated proteolysis of Rab32:GTP (red). GTP-hydrolysis was monitored by quantifying the GTP-content using ion-pairing reversed-phase chromatography (Supplementary Fig. 2A). The proteolysis was monitored by Coomassie-stained SDS-PAGE (Supplementary Fig. 2B). Both data sets are presented as means of technical replicates ( ± SEM; n = 2). The data were fitted to a single exponential function, yielding the observed rate constants k _obs,prot and k _hydr. d Proteolytic cleavage does not promote unfolding of Rab32. Comparison of the retention times of Rab32_FL (8 µM, black) and cleaved Rab32 (8 µM, red) by size-exclusion chromatography (SEC). Inset: Full conversion was verified by SDS-PAGE. e Rab32 cleavage mildly affects thermal stability. Melting point (T _m) determination of Rab32_FL (black) and cleaved Rab32 (red) by differential scanning fluorimetry (DSF). The data were fitted to a Boltzmann equation, yielding the melting temperature. Inset: Results are presented as bar graphs with means ± SEM (n = 3)