PMC:5026484 / 13148-15873 JSONTXT

{"project":"2_test","denotations":[{"id":"27552051-20573983-26907292","span":{"begin":1000,"end":1004},"obj":"20573983"}],"text":"EHBPs colocalize with Rab8-family members\nInterestingly, in addition to the RBDs, EHBPs also contain CaaX-boxes at their C-termini (EHBP1: CVLQ; EHBP1L1: CVLS; Figure 1) for posttranslational modification with prenyl-groups. Therefore, we set out to test whether these motifs can be prenylated in vitro and if prenylation is responsible and necessary for correct intracellular localization. In vitro, both proteins can be farnesylated and geranylgeranylated by FTase and GGTase I, respectively. However, in accordance with the C-terminal amino acids of the CaaX-boxes being glutamine or serine (Zhang and Casey, 1996), we observed preferential farnesylation (Figure 3a). Using constructs containing the RBD with or without the CaaX-boxes, we next looked at the intracellular localization. These experiments clearly showed a CaaX-box dependent localization of both proteins to intracellular structures resembling endosomes (Figure 3b), as previously reported for the full length proteins (Shi et al., 2010). Additionally, both proteins showed strong colocalization with constitutively active Rab8 and Rab10 (i.e. Rab8Q67L and Rab10Q68L), both known to act at endosomes (Figure 3c), further supporting the function of this family of effector proteins as Rab8-family binding partners.\n10.7554/eLife.18675.008Figure 3. Prenylated EHBP1 and EHBP1L1 colocalize with Rab8 and Rab10.\n(a) EHBP1 and EHBP1L1 can be prenylated in vitro as shown by mass spectrometry. After incubation of the purified proteins including the CaaX-motifs (theoretical masses of the purified proteins: 22253.3 Da (EHBP1); 22214.0 Da (EHBP1L1); left panel) with Farnesytransferase (FTase, middle panel) or Geranylgeranyltransferase (GGTase, right panel) farnesylation/geranylgeranylation lead to an increase in mass of 205.4/272.5 Da, respectively. Note that farnesylation, in contrast to geranylgeranylation, appears to be more efficient under similar conditions and goes to completion. This is in agreement with the sequence of the CaaX-motifs in both proteins containing a Gln/Ser at their C-terminus which has been shown to favor farnesylation. (b) Whereas the constructs containing the bMERB domain and the CaaX-motif (EHBP11047-1231, EHBP1L11340-1523) localize to intracellular structures resembling endosomes, deletion of the CaaX-motif (∆CaaX) leads to a cytosolic distribution for both EHBP1 and EHBP1L1 (Scale bars: 10 µm). (c) Both EGFP-EHBP11047-1231 and EGFP-EHBP1L11340-1523 (upper panel) show strong colocalisation with mCherry-Rab8Q67L and mCherry-Rab10Q68L (middle panel) as indicated in the merged images (lower panel). The localization pattern resembles that of endosomes (Scale bars: 10 µm).\nDOI: http://dx.doi.org/10.7554/eLife.18675.008"}

{"project":"MyTest","denotations":[{"id":"27552051-20573983-26907292","span":{"begin":1000,"end":1004},"obj":"20573983"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"EHBPs colocalize with Rab8-family members\nInterestingly, in addition to the RBDs, EHBPs also contain CaaX-boxes at their C-termini (EHBP1: CVLQ; EHBP1L1: CVLS; Figure 1) for posttranslational modification with prenyl-groups. Therefore, we set out to test whether these motifs can be prenylated in vitro and if prenylation is responsible and necessary for correct intracellular localization. In vitro, both proteins can be farnesylated and geranylgeranylated by FTase and GGTase I, respectively. However, in accordance with the C-terminal amino acids of the CaaX-boxes being glutamine or serine (Zhang and Casey, 1996), we observed preferential farnesylation (Figure 3a). Using constructs containing the RBD with or without the CaaX-boxes, we next looked at the intracellular localization. These experiments clearly showed a CaaX-box dependent localization of both proteins to intracellular structures resembling endosomes (Figure 3b), as previously reported for the full length proteins (Shi et al., 2010). Additionally, both proteins showed strong colocalization with constitutively active Rab8 and Rab10 (i.e. Rab8Q67L and Rab10Q68L), both known to act at endosomes (Figure 3c), further supporting the function of this family of effector proteins as Rab8-family binding partners.\n10.7554/eLife.18675.008Figure 3. Prenylated EHBP1 and EHBP1L1 colocalize with Rab8 and Rab10.\n(a) EHBP1 and EHBP1L1 can be prenylated in vitro as shown by mass spectrometry. After incubation of the purified proteins including the CaaX-motifs (theoretical masses of the purified proteins: 22253.3 Da (EHBP1); 22214.0 Da (EHBP1L1); left panel) with Farnesytransferase (FTase, middle panel) or Geranylgeranyltransferase (GGTase, right panel) farnesylation/geranylgeranylation lead to an increase in mass of 205.4/272.5 Da, respectively. Note that farnesylation, in contrast to geranylgeranylation, appears to be more efficient under similar conditions and goes to completion. This is in agreement with the sequence of the CaaX-motifs in both proteins containing a Gln/Ser at their C-terminus which has been shown to favor farnesylation. (b) Whereas the constructs containing the bMERB domain and the CaaX-motif (EHBP11047-1231, EHBP1L11340-1523) localize to intracellular structures resembling endosomes, deletion of the CaaX-motif (∆CaaX) leads to a cytosolic distribution for both EHBP1 and EHBP1L1 (Scale bars: 10 µm). (c) Both EGFP-EHBP11047-1231 and EGFP-EHBP1L11340-1523 (upper panel) show strong colocalisation with mCherry-Rab8Q67L and mCherry-Rab10Q68L (middle panel) as indicated in the merged images (lower panel). The localization pattern resembles that of endosomes (Scale bars: 10 µm).\nDOI: http://dx.doi.org/10.7554/eLife.18675.008"}