1. GORAB Missense Mutations Disrupt RAB6 and ARF5 Binding and Golgi Targeting 
Johannes Egerer, Denise Emmerich, Björn Fischer-Zirnsak, Wing Lee Chan, David Meierhofer, Beyhan Tuysuz, Katrin Marschner, Sascha Sauer, Francis A. Barr, Stefan Mundlos, Uwe Kornak 
Journal of Investigative Dermatology 
Volume 135, Issue 10, Pages (October 2015)
DOI: /jid
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2. Figure 1
Golgin, RAB6-interacting (GORAB) localizes at the trans-Golgi and is redistributed to the ER by Brefeldin A treatment. (a) HeLa cells or (b) human skin fibroblasts were treated for 3 h with 33 μM Nocodazole to produce Golgi ministacks. Costaining for GORAB (red), and the subcellular markers GM130 (cis-Golgi), p230 (trans-Golgi), and TGN46 (trans-Golgi network; green) show best overlap of GORAB with the trans-Golgi markers. Lower images show magnification of area marked by white boxes. White bars indicate areas of confocal colocalization measurements shown in the lowest panel. Traces evaluate the degree of colocalization. (c) HeLa cells were treated for the indicated times with 5 μg ml−1 Brefeldin A blocking ER-Golgi transport. GORAB (green) was costained with COPI component δCOP, Golgi matrix protein GM130, and Golgi SNARE GS15 (all in red), respectively, by immunofluorescence to monitor the response to the treatment. Scale bars=10 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
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3. Figure 2
Golgin, RAB6-interacting (GORAB) interacts with RAB6 by amino acids 99 to 277. (a) GORAB Fragments used for the mapping of interactions. (b) Yeast 2-hybrid mapping of the interaction between active RAB6 p.Gln72Ala as bait and the indicated fragments of GORAB as prey. Growth control shows the presence of bait and prey constructs in the selected clones. Growth on selective plates shows interaction of bait and prey fusion proteins. (c–f) For in vitro pulldown experiments, wild-type, guanosine triphosphate (GTP)-loaded RAB6 (RAB6 GTP) or inactive RAB6 p.Thr27Asn (RAB6 n.act.) were expressed as glutathione S-transferase (GST) fusion proteins and incubated with lysates from HeLa cells transiently transfected with the indicated FLAG-tagged GORAB fragments. The bound protein was analyzed on western blot with a FLAG-specific antibody. CC, coiled-coil.
Journal of Investigative Dermatology  , DOI: ( /jid )
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4. Figure 3
Golgin, RAB6-interacting (GORAB) interacts specifically with ARF5 by amino acids 99 to 277. (a) Yeast 2-hybrid screening for GORAB interaction partners from the ARF family. The growth control shows the presence of bait and prey constructs, and growth on selective plates indicates interaction of bait and prey fusion proteins. (b) In vitro pulldown experiment with guanosine triphosphate (GTP)-locked ARF5 p.Gln71Leu (ARF5 active) and ARF4 p.Gln71Leu (ARF4 active) or GDP-loaded, wild-type ARF5 (ARF5 GDP). The glutathione S-transferase (GST) fusion proteins were incubated with lysates from HeLa cells, and bound endogenous GORAB protein was detected by western blotting. (c) Colocalization of HeLa cells transfected with GFP-tagged, active ARF5 (p.Gln71Leu) and costained for endogenous GORAB (red). (d) Interaction of wild-type ARF5 as bait and the indicated fragments of GORAB as prey was mapped by yeast 2-hybrid. (e) In vitro pulldown experiments were performed with ARF5 p.Gln71Leu (ARF5 active) or wild-type ARF5 loaded with either GTP (ARF5 GTP) or GDP (ARF5 GDP). FLAG-tagged full-length human GORAB was expressed in HeLa cells, and bound protein was analyzed by western blotting. (f–h) In vitro pulldown mapping was performed as in d, e but only ARF5 GTP and ARF5 GDP were used. Scale bars=10 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
The Golgi-targeting motif of golgin, RAB6-interacting (GORAB) is found within amino acids 200 to 277 of the internal Golgi-targeting Rab6 and Arf5 binding domain (IGRAB) domain. Confocal images of HeLa cells transfected transiently with the indicated FLAG-tagged GORAB fragments (green) and costained for the cis-Golgi markers GM130 or giantin (red) show targeting of the respective fragments. The minimal fragment still partially targeting to the Golgi compartment comprises amino acids 200 to 277. Scale bars=10 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Missense mutations in the internal Golgi-targeting Rab6 and Arf5 binding domain (IGRAB) domain disturb ARF5 and RAB6 binding and Golgi targeting. (a) Golgin, RAB6-interacting (GORAB) p.Ala220Pro was expressed in HeLa cells and stained for FLAG (green) and the Golgi marker giantin (red). (b) Guanosine triphosphate (GTP)-loaded glutathione S-transferase (GST)-RAB6 (RAB6 GTP) or inactive GST-RAB6 p.Thr27Asn (RAB6 n.act.) was used for pulldown of FLAG-tagged GORAB p.Ala220Pro expressed in HeLa cells. Bound (pulldown) and unbound (supernatant) protein fractions were blotted. (c) Same as in b, but wild-type GST-ARF5 was loaded with either GTP (ARF5 GTP) or GDP (ARF5 GDP) and used for pulldown of GORAB p.Ala220Pro. (d) Skin fibroblasts from a patient with the GORAB p.Ser175Phe mutation were stained for GORAB (red) and the Golgi marker GM130 (green). (e) GTP-loaded GST-RAB6 (RAB6 GTP) or inactive GST-RAB6 p.Thr27Asn (RAB6 n.act.) was used to pulldown FLAG-tagged GORAB p.Ser175Phe expressed in HeLa cells. (f) Same as in b, but GTP-locked GST-ARF5 p.Gln71Leu (ARF5 act.) and GST-ARF5, loaded with either GTP (ARF5 GTP) or GDP (ARF5 GDP), were used for the pulldown of GORAB p.Ser175Phe. Scale bars=10 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions