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. Author manuscript; available in PMC: 2010 Jun 1.
Published in final edited form as: Nat Cell Biol. 2009 Nov 15;11(12):1399–1410. doi: 10.1038/ncb1986

Figure 1.

Figure 1

AMPH-1 physically interacts with RME-1. (a) A flowchart representation of the steps involved in identifying AMPH-1 as an RME-1 EH-domain interacting protein. Bioinformatic searches of the C. elegans proteome identified multi-NPF and NPF(D/E) containing candidates which were assayed for effects on GFP-RME-1 subcellular localization after RNAi-mediated depletion, leading to identification of AMPH-1, a BAR (Bin1-Amphiphysin-Rvs161p/167p) and SH3 (Src-homology domain 3) domain protein. A diagram of the C. elegans amph-1 gene indicating 5’ and 3’ untranslated regions (dark gray boxes), exons (light gray boxes), introns (connecting lines), and the location of the amph-1(tm1060) deletion. (b) RME-1 (residues 447–555) was expressed as bait in a yeast reporter strain. AMPH-1 (residues 230–394) and its mutant forms were expressed as prey in the same yeast cells. Interaction between bait and prey was assayed by quantitative β-galactosidase (β-gal) assays. Mutation of either NPF motif to NPA is sufficient to significantly disrupt the interaction. The y-axis is labeled in Miller units. n=2, data from independent experiments is indicated above the graphs. (c) Glutathione beads loaded with recombinant GST or GST-RME- 1(442–576) were incubated with in vitro-expressed HA-AMPH-1(+) or HA-AMPH-1(F309A, F363A). Western blot for bound proteins was probed with anti-AMPH-1 antibody. Input lanes contain 50% target protein. The lower panel shows equivalent loading of bait GST fusion proteins. (d) Glutathione beads loaded with recombinant GST or GST-AMPH-1(full length) were incubated with in vitro-expressed HA-RME-1(full length). Bound proteins were analyzed by Western blot probed with anti-HA antibody. Input lanes contain 10% of the target protein HA-RME-1. The lower panel shows equivalent loading of bait GST fusion proteins. (e) Glutathione beads loaded with recombinant GST, GST-RME- 1(442–576), GST-mRme1/EHD1(EH domain), GST-Eps15(EH domain 2) or GST-Intersectin(EH domains a+b) were incubated with in vitro-expressed target protein HA-AMPH-1(+). Bound proteins were analyzed by Western blot with anti-AMPH-1 antibody. Input lane contains 10% target protein. The lower panel shows equivalent loading of bait GST fusion proteins. (f) Glutathione beads loaded with recombinant GST or GST-RME- 1(442–576) were incubated with in vitro-expressed target proteins HA-AMPH-1(+) or HA-AMPH-1(D310-312A, D364-366A). Bound proteins were analyzed by western blot with anti-AMPH-1 antibody. Input lanes contain 50% of the target protein. The lower panel shows equivalent loading of bait GST fusions.