Journal: eLife

Article Title: The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

doi: 10.7554/eLife.03648

Figure Lengend Snippet: ( A ) Predicted location of residues stabilizing an important inter-subunit salt bridge within the inactive (PBD ID: 2VGL) crystal structure of the vertebrate AP2 core complex. Alpha is blue, beta is green, and mu2 is pink. The residue numbers are from the worm subunits. See and for localization on interfaces. ( B ) Starvation assay (days required for a worm population to expand and consume the bacterial food). AP2 mutations indicated above. ( C ) Cargo assay (amount of GFP-tagged cargo on intestinal cell membrane). For ( B ) and ( C ), data represent the mean ± SEM for n ≥ 10. Significance determined by unpaired, two-tailed t-test, **p < 0.01. ( D ) in vivo TEV protease assay. Samples collected for western blot analysis (anti-HA) 8 hr after heatshock (As in ). Numbers indicate band intensity normalized to the fcho(+) sample. ( E ) Blot for phosphorylated threonine 160. Samples collected before heatshock. Numbers indicate band intensity normalized to fcho(+) sample. DOI: http://dx.doi.org/10.7554/eLife.03648.013

Article Snippet: Primary antibodies and dilutions include mouse anti-FLAG (1:1000, M2, Sigma-Aldrich, St. Louis, MO), rabbit anti-APM1 (phospho T156, 1:1000, Abcam 109397, Cambridge, England), rabbit anti-histone H3 (1:100000, Abcam 1791), and rabbit-anti TEV protease (1:500, Rockland Immunochemicals, Limerick, PA).

Techniques: Residue, Membrane, Two Tailed Test, In Vivo, Protease Assay, Western Blot

Journal: eLife

Article Title: The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

doi: 10.7554/eLife.03648

Figure Lengend Snippet: ( A ) A TEV protease site was inserted into a surface loop of the mu domain. The dashed line connects the boundaries of the unstructured region within two conformations of the AP2 complex. ( B ) Western blot of whole animal lysates expressing the HA-tagged mu2 subunit depicted in . The amount of full-length subunit (top) decreases following heatshock. Anti-histone blot is below. Each sample is comprised of 100 larval 4 stage animals. ( C ) Quantification of mu2 proteolysis. Intensity of anti-HA signal relative to histone, normalized to time 0. *p < 0.05, unpaired, two-tailed t-test compared to fcho(+) values at same time point, n = 4. Data represent the mean ± SEM. See for results using FLAG-tagged version of mu2. DOI: http://dx.doi.org/10.7554/eLife.03648.009

Article Snippet: Primary antibodies and dilutions include mouse anti-FLAG (1:1000, M2, Sigma-Aldrich, St. Louis, MO), rabbit anti-APM1 (phospho T156, 1:1000, Abcam 109397, Cambridge, England), rabbit anti-histone H3 (1:100000, Abcam 1791), and rabbit-anti TEV protease (1:500, Rockland Immunochemicals, Limerick, PA).

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: eLife

Article Title: The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

doi: 10.7554/eLife.03648

Figure Lengend Snippet: ( A ) Cartoon of modified mu2 including epitope tag, sigma-homology domain, mu domain and TEV protease site insertion. The protease site is predicted to become more accessible upon AP2 activation. ( B ) Western blots of whole worm lysates. Animals were heat-shocked for one hour to induce TEV protease expression. Full-length (intact) and proteolyzed (cleaved) mu2 subunits were detected using anti-FLAG (top) while protease expression was monitored with anti-TEV (middle). Anti-histone blot (bottom) used for normalization. Each sample is comprised of 100 larval 4 stage animals. ( C ) Quantification of the proteolysis. Intensity of intact mu2 relative to histone, normalized to time 0. Similar results were obtained using an HA-tagged mu2 subunit . ( D ) Protease levels over time. Intensity of TEV band relative to histone. DOI: http://dx.doi.org/10.7554/eLife.03648.010

Article Snippet: Primary antibodies and dilutions include mouse anti-FLAG (1:1000, M2, Sigma-Aldrich, St. Louis, MO), rabbit anti-APM1 (phospho T156, 1:1000, Abcam 109397, Cambridge, England), rabbit anti-histone H3 (1:100000, Abcam 1791), and rabbit-anti TEV protease (1:500, Rockland Immunochemicals, Limerick, PA).

Techniques: Modification, Activation Assay, Western Blot, Expressing

Journal: eLife

Article Title: The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

doi: 10.7554/eLife.03648

Figure Lengend Snippet: Listed mutations correspond to the worm residues. ( A ) Starvation assay (days required for a worm population to expand and consume the bacterial food). ( B ) Cargo assay (amount of GFP-tagged cargo on intestinal cell membrane). For ( A ) and ( B ), data represent the mean ± SEM for n ≥ 10. Significance determined by unpaired, two-tailed t-test compared to fcho(−), *p < 0.05 and **p < 0.01. ( C ) in vivo TEV protease assay. Samples collected for Western blot analysis 8 hr after heatshock (as in ). Numbers indicate band intensity of full-length mu2 (anti-HA, top) relative to histone (bottom), normalized to the fcho(+) sample. ( D ) Blot for phosphorylated threonine-160 (T156 in vertebrates) in the linker region of the mu2 subunit. Samples collected before heatshock. Numbers indicate band intensity of phosphorylated T160 (top) relative to histone (bottom), normalized to fcho(+) sample. For ( C ) and ( D ), each sample is comprised of 100 larval 4 stage animals. DOI: http://dx.doi.org/10.7554/eLife.03648.011

Article Snippet: Primary antibodies and dilutions include mouse anti-FLAG (1:1000, M2, Sigma-Aldrich, St. Louis, MO), rabbit anti-APM1 (phospho T156, 1:1000, Abcam 109397, Cambridge, England), rabbit anti-histone H3 (1:100000, Abcam 1791), and rabbit-anti TEV protease (1:500, Rockland Immunochemicals, Limerick, PA).

Techniques: Membrane, Two Tailed Test, In Vivo, Protease Assay, Western Blot

Journal: eLife

Article Title: The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

doi: 10.7554/eLife.03648

Figure Lengend Snippet: In ( A ) and ( B ) APA domains from FCHo homologs were expressed as HaloTag fusions in HEK293T cells ( Ce , C. elegans ; Mm , M. musculus ). ( A ) Silver-stained gel of affinity-purified proteins following proteolytic cleavage from the HaloTag. Arrows indicate bands of presumed identity. ( B ) The top ten human proteins purified using two different APA baits, as detected by MudPIT mass spectrometry. Nonspecific proteins also found in the control were removed. Values represent the mean % distributed Normalized Spectral Abundance Factor (dNSAF × 100) from three independent experiments. The values of all four AP2 subunits were totaled to determine the amount of complex in each sample. Multiple isoforms of alpha, beta, and phosphatidylinositol 5-phosphate 4-kinase type-2 (PIP4K2) were summed. WD repeat-containing protein 48 (WDR48), epidermal growth factor receptor substrate 15 (EPS15), adaptin ear-binding coat-associated protein 2 (NECAP2), and serine/threonine-protein kinase PLK1 (PLK1). See for complete results. ( C ) APA pulldowns using bacterially expressed proteins. Purified HaloTag with (HT + APA) and without (HT) the APA domain from mouse SGIP1 were incubated with purified AP2 fragments followed by TEV protease cleavage to release the APA bait. Silver-stained gel of the eluted proteins. Note that the alpha/sigma and beta/mu hemicomplexes are soluble in our hands and that the AP2 appendage (ear) domains exhibit non-specific binding in this assay. DOI: http://dx.doi.org/10.7554/eLife.03648.017

Article Snippet: Primary antibodies and dilutions include mouse anti-FLAG (1:1000, M2, Sigma-Aldrich, St. Louis, MO), rabbit anti-APM1 (phospho T156, 1:1000, Abcam 109397, Cambridge, England), rabbit anti-histone H3 (1:100000, Abcam 1791), and rabbit-anti TEV protease (1:500, Rockland Immunochemicals, Limerick, PA).

Techniques: Staining, Affinity Purification, Purification, Mass Spectrometry, Control, Binding Assay, Incubation