(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).
DOI:
http://dx.doi.org/10.7554/eLife.18675.008