1. Cell Biology

NECAPs are negative regulators of the AP2 clathrin adaptor complex

  1. Gwendolyn M Beacham
  2. Edward A Partlow
  3. Jeffrey J Lange
  4. Gunther Hollopeter  Is a corresponding author
  1. Cornell University, United States
  2. Stowers Institute for Medical Research, United States
https://doi.org/10.7554/eLife.32242
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Cite this article
  1. Gwendolyn M Beacham
  2. Edward A Partlow
  3. Jeffrey J Lange
  4. Gunther Hollopeter
(2018)
NECAPs are negative regulators of the AP2 clathrin adaptor complex
eLife 7:e32242.
https://doi.org/10.7554/eLife.32242
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7 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
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Loss of NCAP-1 suppresses fcho-1 mutants.

(A) Gene model of the C. elegans ncap-1 locus. Boxes represent exons. Mutations isolated from the fcho-1 suppressor screen are indicated. The deletion allele, mew39, was used throughout this study as ncap-1. The neighboring gene (Y110A2AR.1) is predicted to encode a receptor expression-enhancing protein (REEP). (B) Animal heads showing jowls phenotype (red arrows). Anterior is up. WT, wild type; RFP:NCAP-1, red fluorescent protein-tagged NCAP-1 single-copy transgene. (C) Starvation assay. Data represent days required for worms to reproduce and consume bacterial food source (top schematic). Bars indicate mean ±SEM for n = 10 biological replicates.

https://doi.org/10.7554/eLife.32242.002
Figure 1—figure supplement 1
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AP2 structures and mutations.

(A) AP2 is comprised of two large adaptins (α and β2) and two smaller subunits (µ2 and σ2). The adaptins, in turn, are comprised of appendage (ear), hinge (linker) and trunk domains. Phosphorylation site and binding pockets are diagrammed. (B) Cartoon representations of the closed (Collins et al., 2002) and open (Jackson et al., 2010 and Kelly et al., 2014) AP2 conformations. The core complex (dashed line) lacks ears and linkers. (C) Table of AP2 mutations used in this study.

https://doi.org/10.7554/eLife.32242.003
Figure 2
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Loss of NCAP-1 restores AP2 activity in fcho-1 mutants.

(A) FRAP analysis of GFP-tagged AP2 α adaptin (APA-2:GFP) on membranes of coelomocytes (top schematic). Time constants (tau) of the fluorescence recovery are plotted. (B) AP2 localization in coelomocytes. Representative confocal images of coelomocytes in worms expressing APA-2:GFP. Micrographs (top) are representative maximum projections of Z-slices through approximately half of a cell. Data represent the coefficient of variance (%CV) of pixel intensities for individual cells. (C) Artificial AP2 cargo assay. Representative confocal micrographs of intestinal cells (middle) in worms expressing a GFP-tagged cargo (top schematic). TM, transmembrane domain. The average pixel intensity along a basolateral membrane was measured (bottom). (A–C) Bars indicate mean ±SEM for n ≥ 8 biological replicates. *p<0.05, **p<0.001, not significant (n.s.), unpaired, two-tailed T-test. (D) µ2 protease-sensitivity assay. Western blot analysis of whole worm lysates was used to quantify the amount of full-length µ2 (anti-HA, 50 kDa) before (pre TEV, bottom blot) and after protease induction (post TEV, top blot). Band intensities were compared to a tubulin loading control and normalized to the fcho-1(+) ncap-1(+) ratio (values below). (E) µ2 phosphorylation assay. Western blot analysis of whole worm lysates to quantify phosphorylated µ2 (top blot) relative to total µ2 subunit (bottom blot). Values indicate band intensity ratios of phospho µ2 compared to total µ2, normalized to the fcho-1(+) ncap-1(+) ratio (values below). (D and E) Blots are representative of ≥3 biological replicates. +, wild type allele; -, deletion allele.

https://doi.org/10.7554/eLife.32242.004
Figure 3
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NECAPs restore closed AP2 in fcho-1 ncap-1 worms.

RFP-tagged NECAPs were expressed as single copy transgenes in fcho-1 ncap-1 worms. Ce, C. elegans; Mm, M. musculus; Ss, Sphaerobolus stellatus (multicellular fungus). +, wild type allele; -, deletion allele. (A) Starvation assay performed as in Figure 1C. Bars represent mean ±SEM for n ≥ 7 biological replicates. **p<0.001, unpaired, two-tailed T-test. (B) µ2 protease-sensitivity assay as in Figure 2D, except a flag-tagged µ2 subunit was used. Band intensities were compared to a histone loading control and normalized to the fcho-1(+) ncap-1(+) ratio (values below). Blot is representative of 2 biological replicates.

https://doi.org/10.7554/eLife.32242.005
Figure 4
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NECAPs bind the open and phosphorylated AP2 core.

Pulldown assays using affinity-tagged NECAPs. Proteins were cleaved from the affinity tag (HaloTag), electrophoretically separated and then blotted for AP2 subunits (A and C) or SYPRO-stained prior to imaging (B). Control, HaloTag alone; NC, NECAP; Ce, C. elegans; Mm, M. musculus. (A) Western blot analysis (middle) of samples purified from human cell lysates (top schematic) expressing the indicated NECAP bait (bottom). (B and C) In vitro pulldown assays using purified recombinant bait (NECAPs, bottom) and prey (vertebrate AP2 cores, top). Co-expression with the kinase domain from mouse AAK1 (+kinase) generates phosphorylated AP2. Amino acid changes in µ2 are indicated: E302K, constitutively open AP2; T156A, phosphorylation-defective AP2; see also Figure 1—figure supplement 1C. 20% of prey input was analyzed for comparison with 50% of the sample released by the protease. (A–C) Band intensities of the α subunit (A) or the α trunk (B and C) were quantified, background signal subtracted, and values normalized to the HaloTag control (values above). Data are representative of 2 biological (A), one technical (B), and two technical (C) replicates.

https://doi.org/10.7554/eLife.32242.006
Figure 5
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Phosphorylated AP2 recruits NCAP-1 in vivo.

Representative confocal slices (middle) through the approximate center of the nerve ring of worms (top schematic) expressing RFP:NCAP-1 and APA-2:GFP. RFP to GFP signal intensity at the nerve ring is plotted (bottom). Mutations in µ2 are indicated: E306K and R440S, constitutively open AP2; T160A, phosphorylation-defective AP2; see also Figure 1—figure supplement 1C. Bars indicate mean ±SEM for n ≥ 10 biological replicates. **p<0.001, unpaired, two-tailed T-test. au, arbitrary units; +, wild type allele; -, deletion allele.

https://doi.org/10.7554/eLife.32242.007
Figure 6 with 1 supplement
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Missense mutations render NCAP-1 stable but functionally inactive.

Amino acid changes isolated from the fcho-1 suppressor screen (Figure 1A) were introduced into an RFP-tagged NCAP-1 transgene in fcho-1 ncap-1 worms. +, wild type allele; -, deletion allele. (A) Starvation assay performed as in Figure 1C. Bars represent mean ±SEM for n ≥ 9 biological replicates. **p<0.001, unpaired, two-tailed T-test. (B) µ2 protease-sensitivity assay as in Figure 3B. (C) Western blot analysis to detect HA epitope on NCAP-1 transgenic proteins. (B and C) Band intensities were compared to a beta actin loading control and normalized to the fcho-1(+) ncap-1(+) ratio (B) or to the transgenic wild type form of NCAP-1 (C) (values below). Blots are representative of 2 biological replicates.

https://doi.org/10.7554/eLife.32242.008
Figure 6—figure supplement 1
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Missense mutations in NECAPs prevent association with phosphorylated forms of AP2.

(A) Confocal slices of worm nerve rings were acquired and analyzed as for Figure 5. Amino acid changes in µ2 (E306K, constitutively open AP2; see also Figure 1—figure supplement 1C) and in NCAP-1 are indicated. Bars indicate mean ±SEM for n = 10 biological replicates. **p<0.001, unpaired, two-tailed T-test. au, arbitrary units; +, wild type allele; -, deletion allele. (B) In vitro pulldown assays were performed as described in Figure 4B. Missense mutations in Mm NECAP2 bait are indicated (below). +, wild type allele. Data are representative of 1 technical replicate. Band intensities of the α trunk were quantified as in Figure 4 (values above).

https://doi.org/10.7554/eLife.32242.009
Figure 7
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Model of AP2 activation and inactivation.

Muniscins allosterically activate AP2 to form a stable association with the membrane. Open AP2 is then phosphorylated on the µ2 subunit by the AP2-associated kinase. NECAPs subsequently bind to open, phosphorylated AP2 and recycle the complex. Generation of the closed form of AP2 presumably involves dephosphorylation and disengagement from the membrane. In the absence of muniscins, AP2 activation is greatly reduced. The fcho-1 suppressor screen isolated three classes of mutations that enable AP2 to remain active in lieu of muniscins (bottom). Each class disrupts the AP2 inactivation pathway and promotes accumulation of AP2 at discrete steps in the cycle (gray arrows).

https://doi.org/10.7554/eLife.32242.010

Tables

Key resources table
Reagent type (species)
or resource		DesignationSource or referenceIdentifiersAdditional information
gene (Caenorhabditis
elegans)		ncap-1NACELE_Y110A2AR.3
gene (C. elegans)fcho-1NACELE_F56D12.6
gene (C. elegans)apm-2NACEAP50, apm-2,
CELE_R160.1
gene (C. elegans)apa-2NAapt-4, CELE_T20B5.1
strain, strain background
(C. elegans, hermaphrodite)		N2NARRID:WB-STRAIN:N2_(ancestral))Wild type
strain, strain background
(C. elegans, hermaphrodite)		GUN109this paperfcho-1(ox477::unc-119(+)) II
ncap-1(mew31[splice donor]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN110this paperfcho-1(ox477::unc-119(+)) II
ncap-1(mew32[A29D]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN111this paperfcho-1(ox477::unc-119(+)) II
ncap-1(mew33[S84N]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN112this paperfcho-1(ox477::unc-119(+)) II
ncap-1(mew34[splice donor]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN113this paperfcho-1(ox477::unc-119(+)) II
ncap-1(mew35[splice donor]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN114this paperfcho-1(ox477::unc-119(+)) II
ncap-1(mew36[Q107X]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN115this paperfcho-1(ox477::unc-119(+)) II
ncap-1(mew37[splice donor]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN116this paperfcho-1(ox477::unc-119(+)) II
ncap-1(mew38[stop lost]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN101this paperfcho-1(ox477::unc-119(+)) II
ncap-1(mew39[1.4 kb deletion]) II
strain, strain background
(C. elegans, hermaphrodite)		EG6353DOI: 10.7554/eLife.03648fcho-1(ox477::unc-119(+)) II;
unc-119(ed3) III
strain, strain background
(C. elegans, hermaphrodite)		GUN86this paperncap-1(mew39[1.4 kb deletion]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN59this paperfcho-1(ox477::unc-119(+)) II mewSi2
[Pdpy-30::RFP:NCAP1 unc-119(+)] II
ncap-1(mew39[1.4 kb deletion]) II
strain, strain background
(C. elegans, hermaphrodite)		EG8012DOI: 10.7554/eLife.03648oxSi254[Pdpy-30::APA-2::GFP
unc-119(+)] II; unc-119(ed3) III
strain, strain background
(C. elegans, hermaphrodite)		EG6650DOI: 10.7554/eLife.03648fcho-1(ox477::unc-119(+)) II oxSi254
[Pdpy-30::APA-2::GFP unc-119(+)] II
strain, strain background
(C. elegans, hermaphrodite)		GUN98this paperfcho-1(ox477::unc-119(+)) II oxSi254
[Pdpy-30::APA-2::GFP unc-119(+)] II
ncap-1(mew39[1.4 kb deletion]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN97this paperoxSi254[Pdpy-30::APA-2::GFP
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II
strain, strain background
(C. elegans, hermaphrodite)		EG8578DOI: 10.7554/eLife.03648oxSi484[Pvha-6::GFP:CD4:YASV
unc-119(+)] II; apm-2(ox546[W64X]) X
oxSi876[Papm-2::HA:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		EG8579DOI: 10.7554/eLife.03648fcho-1(ox477::unc-119(+)) II
oxSi484[Pvha-6::GFP:CD4:YASV
unc-119(+)] II; apm-2(ox546[W64X]) X
oxSi876[Papm-2::HA:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN65this paperfcho-1(ox477::unc-119(+)) II
oxSi484[Pvha-6::GFP:CD4:YASV
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II; apm-2(ox546[W64X]) X
oxSi876[Papm-2::HA:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN66this paperoxSi484[Pvha-6::GFP:CD4:YASV
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II;
apm-2(ox546[W64X]) X oxSi876
[Papm-2::HA:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		EG8557DOI: 10.7554/eLife.03648oxSi883[Phsp-16.41::TEV(protease)
unc-119(+)] II; apm-2(ox546[W64X]) X
oxSi876[Papm-2::HA:APM-2:
tev-site unc-119(+)]
strain, strain background
(C. elegans, hermaphrodite)		EG8558DOI: 10.7554/eLife.03648fcho-1(ox477::unc-119(+)) II
oxSi883[Phsp-16.41::TEV(protease)
unc-119(+)] II; apm-2(ox546[W64X]) X
oxSi876[Papm-2::HA:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN100this paperfcho-1(ox477::unc-119(+)) II;
oxSi883[Phsp-16.41::TEVprotease
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II; apm-2(ox546[W64X]) X
oxSi876[Papm-2::HA:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN99this paperoxSi883[Phsp-16.41::TEVprotease
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II; apm-2(ox546[W64X]) X
oxSi876[Papm-2::HA:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		EG8555DOI: 10.7554/eLife.03648oxSi883[Phsp-16.41::TEVprotease
unc-119(+)] II; apm-2(ox546[W64X]) X
oxSi877[Papm-2::3XFLAG:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		EG8556DOI: 10.7554/eLife.03648fcho-1(ox477::unc-119(+)) II
oxSi883[Phsp-16.41::TEVprotease
unc-119(+)] II; apm-2(ox546[W64X]) X
oxSi877[Papm-2::3xFLAG:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN96this paperfcho-1(ox477::unc-119(+)) II
oxSi883[Phsp-16.41::TEVprotease
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II; apm-2(ox546[W64X]) X
oxSi877[Papm-2::3xFLAG:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN106this paperfcho-1(ox477::unc-119(+)) II
oxSi883[Phsp-16.41::TEVprotease
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II; mewSi3
[Pdpy-30::RFP:NCAP-1 unc-119(+)] IV;
apm-2(ox546[W64X]) X oxSi877
[Papm-2::3xFLAG:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN91this paperfcho-1(ox477::unc-119(+)) II
oxSi883[Phsp-16.41::TEVprotease
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II; mewSi15
[Pdpy-30::RFP:Mm_NECAP1
unc-119(+)] IV; apm-2(ox546[W64X]) X
oxSi877[Papm-2::3xFLAG:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN93this paperfcho-1(ox477::unc-119(+)) II
oxSi883[Phsp-16.41::TEVprotease
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II; mewSi8
[Pdpy-30::RFP:Mm_NECAP2
unc-119(+)] IV; apm-2(ox546[W64X]) X
oxSi877[Papm-2::3xFLAG:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN95this paperfcho-1(ox477::unc-119(+)) II
oxSi883[Phsp-16.41::TEVprotease
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II; mewSi17
[Pdpy-30::RFP:Ss_NECAP
unc-119(+)] IV;
apm-2(ox546[W64X]) X oxSi877
[Papm-2::3xFLAG:APM-2:
tev-site unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN60this papermewSi1[Pdpy-30::APA2:GFP
unc-119(+)] I; mewSi2[Pdpy-30::
RFP:NCAP1 unc-119(+)] II
ncap-1(mew39[1.4 kb deletion]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN61this papermewSi1[Pdpy-30::APA2:GFP
unc-119(+)] I; fcho-1(ox477::
unc-119(+)) II mewSi2[Pdpy-30::
RFP:NCAP1 unc-119(+)] II
ncap-1(mew39[1.4 kb deletion]) II
strain, strain background
(C. elegans, hermaphrodite)		GUN62this papermewSi1[Pdpy-30::APA2:GFP
unc-119(+)] I; fcho-1(ox477::unc-119(+))
II mewSi2[Pdpy-30::RFP:NCAP1
unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II;
apm-2(ox562[E306K]) X
strain, strain background
(C. elegans, hermaphrodite)		GUN53this papermewSi1[Pdpy-30::APA2:GFP
unc-119(+)] I; fcho-1(ox477::
unc-119(+)) II mewSi2[Pdpy-30::
RFP:NCAP1 unc-119(+)] II
ncap-1(mew39[1.4 kb deletion]) II;
apm-2(mew44[T160A]) X
strain, strain background
(C. elegans, hermaphrodite)		GUN55this papermewSi1[Pdpy-30::APA2:GFP
unc-119(+)] I; fcho-1(ox477::
unc-119(+)) II mewSi2[Pdpy-30::
RFP:NCAP1 unc-119(+)] II
ncap-1(mew39[1.4 kb deletion]) II;
apm-2(ox562[E306K]+mew46[T160A]*) X
strain, strain background
(C. elegans, hermaphrodite)		GUN56this papermewSi1[Pdpy-30::APA2:GFP
unc-119(+)] I; fcho-1(ox477::
unc-119(+)) II mewSi2[Pdpy-30::
RFP:NCAP1 unc-119(+)] II
ncap-1(mew39[1.4 kb deletion]) II;
apm-2(ox562[E306K]+mew47[R440S]*) X
strain, strain background
(C. elegans, hermaphrodite)		GUN128this paperfcho-1(ox477::cb-unc-119(+)) II
oxSi883[Phsp-16.41::TEVprotease
Cb_unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II;
mewSi25[RFP:NCAP-1(A29D)*]
IV; apm-2(ox546[W64X]) X
oxSi877[Papm-2::3xFLAG:APM-2:
tev-site Cb_unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN135this paperfcho-1(ox477::cb-unc-119(+)) II
oxSi883[Phsp-16.41::TEVprotease
Cb_unc-119(+)] II ncap-1(mew39
[1.4 kb deletion]) II;
mewSi35[RFP:NCAP-1(S84N)*] IV;
apm-2(ox546[W64X]) X oxSi877
[Papm-2::3xFLAG:APM-2:
tev-site Cb_unc-119(+)] X
strain, strain background
(C. elegans, hermaphrodite)		GUN127this papermewSi1[APA2:GFP] I; fcho-1(ox477::
unc-119(+)) II mewSi24[RFP:
NCAP1(A29D)*] II ncap-1(mew39[1.4 kb
deletion]) II; apm-2(ox562[E306K])X
strain, strain background
(C. elegans, hermaphrodite)		GUN122this papermewSi1[APA2:GFP] I; fcho-1(ox477::
unc-119(+)) II mewSi31[RFP:
NCAP1(S84N)*] II ncap-1(mew39[1.4 kb
deletion]) II; apm-2(ox562[E306K])X
genetic reagent (C. elegans)fcho-1(ox477::unc-119(+))DOI: 10.7554/eLife.03648
genetic reagent (C. elegans)ncap-1(mew31[splice donor])this paperfcho-1 suppressor
genetic reagent (C. elegans)ncap-1(mew32[A29D])this paperfcho-1 suppressor
genetic reagent (C. elegans)ncap-1(mew33[S84N])this paperfcho-1 suppressor
genetic reagent (C. elegans)ncap-1(mew34[splice donor])this paperfcho-1 suppressor
genetic reagent (C. elegans)ncap-1(mew35[splice donor])this paperfcho-1 suppressor
genetic reagent (C. elegans)ncap-1(mew36[Q107X])this paperfcho-1 suppressor
genetic reagent (C. elegans)ncap-1(mew37[splice donor])this paperfcho-1 suppressor
genetic reagent (C. elegans)ncap-1(mew38[stop lost])this paperfcho-1 suppressor
genetic reagent (C. elegans)ncap-1(mew39[1.4 kb
deletion])		this paperfcho-1 suppressor
genetic reagent (C. elegans)mewSi2[Pdpy-30::RFP:NCAP1
unc-119(+)]		this paperGenerated with MosSCI
genetic reagent (C. elegans)mewSi3[Pdpy-30::RFP:NCAP-1
unc-119(+)]		this paperGenerated with MosSCI
genetic reagent (C. elegans)mewSi15[Pdpy-30::RFP:
Mm_NECAP1 unc-119(+)]		this paperGenerated with MosSCI
genetic reagent (C. elegans)mewSi8[Pdpy-30::RFP:
Mm_NECAP2 unc-119(+)]		this paperGenerated with MosSCI
genetic reagent (C. elegans)mewSi17[Pdpy-30::RFP:
Ss_NECAP unc-119(+)]		this paperGenerated with MosSCI
genetic reagent (C. elegans)oxSi254[Pdpy-30::APA-2::
GFP unc-119(+)]		DOI: 10.7554/eLife.03648
genetic reagent (C. elegans)mewSi1[Pdpy-30::
APA-2::GFP unc-119(+)]		this paperGenerated with MosSCI
genetic reagent (C. elegans)oxSi484[Pvha-6::GFP:CD4:
YASV unc-119(+)]		DOI: 10.7554/eLife.03648
genetic reagent (C. elegans)oxSi883[Phsp-16.41::
TEV(protease) unc-119(+)]		DOI: 10.7554/eLife.03648
genetic reagent (C. elegans)oxSi876[Papm-2::HA:APM-2:
tev-site unc-119(+)]		DOI: 10.7554/eLife.03648
genetic reagent (C. elegans)oxSi880[Papm-2::HA:APM-2(E306K):
tev-site unc-119(+)]		DOI: 10.7554/eLife.03648
genetic reagent (C. elegans)oxSi878[Papm-2::HA:APM-2(T160A):
tev-site unc-119(+)]		DOI: 10.7554/eLife.03648
genetic reagent (C. elegans)oxSi877[Papm-2::3xFLAG:
APM-2:tev-site unc-119(+)]		DOI: 10.7554/eLife.03648
genetic reagent (C. elegans)apm-2(ox562[E306K]
+mew46[T160A])		this papermew46[T160A] generated
by CRISPR
genetic reagent (C. elegans)apm-2(ox562[E306K]
+mew47[R440S])		this papermew47[R440S] generated
by CRISPR
genetic reagent (C. elegans)mewSi25[RFP:NCAP-1(A29D]this paperGenerated by CRISPR
genetic reagent (C. elegans)mewSi35[RFP:NCAP-1(S84N)]this paperGenerated by CRISPR
genetic reagent (C. elegans)mewSi24[RFP:NCAP1(A29D)]this paperGenerated by CRISPR
genetic reagent (C. elegans)mewSi31[RFP:NCAP1(S84N)]this paperGenerated by CRISPR
cell line (Homo
sapiens, female)		HEK293ATCCRRID:CVCL_0045
transfected construct
(H. sapiens)		pGH500, in HEK239 cellsthis paperCloning described in ‘Tissue
culture pulldowns’
transfected construct
(H. sapiens)		pGH501, in HEK239 cellsthis paperCloning described in ‘Tissue
culture pulldowns’
transfected construct
(H. sapiens)		pGH502, in HEK239 cellsthis paperCloning described in ‘Tissue
culture pulldowns’
antibodymouse monoclonal
anti-adaptin α		BD BiosciencesCat# 610501,
RRID:AB_397867		(1:500)
antibodyrabbit polyclonal anti-AP2B1Abcam151961, RRID:
AB_2721072		(1:1000)
antibodyrabbit monoclonal
anti-AP2M1 phospho T156		AbcamCat# 109397,
RRID:AB_10866362		(1:1000)
antibodyrabbit monoclonal anti-AP2S1AbcamCat# 128950,
RRID:AB_11140842		(1:4000)
antibodymouse monoclonal anti-flagSigma-AldrichCat# F3165,
RRID:AB_259529		(1:1000)
antibodymouse monoclonal anti-tubulinSigma-AldrichCat# T5168,
RRID:AB_477579		(1:2000)
antibodyrabbit polyclonal anti-histone H3AbcamCat# 1791,
RRID:AB_302613		(1:4000)
antibodyrabbit polyclonal anti-beta actinAbcamCat# 8227,
RRID:AB_2305186		(1:1000)
antibodygoat anti-mouse Alexa Fluor 488Life TechnologiesCat # A11029,
RRID:AB_2534088		(1:4000)
antibodygoat anti-rabbit Alexa Fluor 647Life TechnologiesCat# A21244,
RRID:AB_10562581		(1:2000)
antibodygoat anti-rabbit
StarBright Blue 700		BioRadCat# 12004161,
RRID: AB_2721073		(1:5000)
antibodygoat anti-mouse IRDye 800CWLI-CORCat# 925–32210,
RRID:AB_2687825		(1:20000)
antibodyrat monoclonal anti-HA-
Horseradish
peroxidase (HRP)		RocheCat# 12013819001
RRID:AB_390917		(1:500)
recombinant DNA reagentpEP29this paperCloning described in ‘C. elegans
NECAP transgenes’
recombinant DNA reagentpEP41this paperCloning described in ‘C. elegans
NECAP transgenes’
recombinant DNA reagentpEP58this paperCloning described in ‘C. elegans
NECAP transgenes’
recombinant DNA reagentpEP71this paperCloning described in ‘C. elegans
NECAP transgenes’
recombinant DNA reagentpGH495this paperCloning described in ‘C. elegans
NECAP transgenes’
recombinant DNA reagentpGH505this paperCloning described in ‘C. elegans
NECAP transgenes’
recombinant DNA reagentpGB19this paperCloning described in
‘Recombinant AP2 cores’
recombinant DNA reagentpGB21this paperCloning described in
‘Recombinant AP2 cores’
recombinant DNA reagentpGB27this paperCloning described in
‘Recombinant NECAPs’
recombinant DNA reagentpGB28this paperCloning described in
‘Recombinant NECAPs’
recombinant DNA reagentpGB29this paperCloning described in
‘Recombinant NECAPs’
recombinant DNA reagentpGB31this paperCloning described in
‘Recombinant AP2 cores’
recombinant DNA reagentpGB81this paperCloning described in
‘Recombinant NECAPs’
recombinant DNA reagentpEP82this paperCloning described in
‘Recombinant AP2 cores’
recombinant DNA reagentpGB91this paperCloning described in
‘Recombinant NECAPs’
recombinant DNA reagentpGB94this paperCloning described in
‘Recombinant NECAPs’
sequence-based reagentpGH494DOI: 10.7554/eLife.03648
sequence-based reagentpGH503this paperCloning described in
‘Recombinant NECAPs’
sequence-based reagentpGH504this paperCloning described in
‘Recombinant AP2 cores’
sequence-based reagentoGH678this paperCGATAGAGAAGGCTTCAACACAC
sequence-based reagentoGH679this paperAGGTATTCAGACATTTTTC
AAATGAAAATCTAC
sequence-based reagentoGH680this paperCAGTCAAAAAATGC
GATAAAAGTACGG
sequence-based reagentoGH681this paperGGACAGGAAATTTC
AATAAATTAGCGATG
sequence-based reagentoEP366this paperAACGGGCGGTAGT
GGAGGCACTGGTATG
GGAGATTACGAGAACGTTTTAATG
sequence-based reagentoEP367this paperTATCACCACTTTGTACAAGAAAGCT
GGGTCTAGAAATCTAATAAA
TTGCCAGACGTCG
sequence-based reagentoEP407this paperGAGGAACGGGCGGTAGTGGAG
GCACTGGTATGGAGGAGAGTG
AGTACGAGTCTGTTCTGT
sequence-based reagentoEP408this paperTCACCACTTTGTACAAGAAAGC
TGGGTCTAGAACTGG
ACCCAGCCGGTG
sequence-based reagentoEP409this paperGGAGGAACGGGCGGTAGTGG
AGGCACTGGTATGGCGGCA
GAGCTGGAATATG
sequence-based reagentoEP410this paperTCACCACTTTGTACAAGAAA
GCTGGGTCTAAAACTGGA
CCCAGTTAGATGGCTGTG
sequence-based reagentoEP391this paperACGTCGTGACTGGGAAAACCC
sequence-based reagentoEP392this paperGCCAGGGTTTTCCCAGTCA
CGACGTTGATCATTGGCA
TGCTGAAATATTC
sequence-based reagentoGH526this paperATGGTTGTGTCGAAAGGCGA
sequence-based reagentoGH528this paperACCAGTGCCTCCACTACCG
CCCGTTCCTCCTGTGCCACC
TTTGTACAGTTCATCCATTCC
sequence-based reagentoGH698this paperGGGGACAAGTTTGTACAAAA
AAGCAGGCTCAAAAATGG
TTGTGTCGAAAGGCGA
sequence-based reagentoGH731this paperGTGACATTAAAGTC
AAAAGCATCTCCTC
sequence-based reagentoGH733this paperGGGGACAAGTTTGTACAAAA
AAGCAGGCTCAAAAATGGGA
GATTACGAGAACGTTTTAAT
sequence-based reagentoGH734this paperGGGGACCACTTTGTACAAGAA
AGCTGGGTTTAGAAATCTAAT
AAATTGCCAGACGTC
sequence-based reagentoGH736this paperGAGGAGATGCTTTTGAC
TTTAATGTCAC
sequence-based reagentoGH738this paperGCGGTAGTGGAGGCACTG
GTATGGGAGATTACGAG
AACGTTTTAATG
sequence-based reagentoGH1011this paperTAGACCCAGCTTTCTTGTA
CAAAGTGGTGATA
sequence-based reagentoGH1012this paperACCAGTGCCTCCAC
TACCGCCCGTT
sequence-based reagentoGH953this paperCATGCTTCCGCCGGTACCT
sequence-based reagentoGH954this paperGTTTAAACCCGCTGATCAGCCT
sequence-based reagentoGH955this paperGTGGAGGTACCGGCGGAAG
CATGGGAGATTACGAG
AACGTTTTAATG
sequence-based reagentoGH956this paperGCTGATCAGCGGGTTTAAACTT
AGAAATCTAATAAATT
GCCAGACGTCG
sequence-based reagentoGH957this paperGTGGAGGTACCGGCGGAA
GCATGGCGGCAGAGCTGGAA
sequence-based reagentoGH958this paperGATCAGCGGGTTTAAACTT
AAAACTGGACCCAGTTAGATGGC
sequence-based reagentoGH959this paperGTGGAGGTACCGGCGGAA
GCATGGAGGAGAGTGAGTACGAGT
sequence-based reagentoGH960this paperGCTGATCAGCGGGTTTAAA
CTTAGAACTGGACCCAGCCGG
sequence-based reagentoEP13this paperTAATTAACCTAGGCTGCTGCCACC
sequence-based reagentoEP17this paperAAGAAGGAGATATACATAT
GAAGAAGTTTTTCGACTCCAG
sequence-based reagentoEP18this paperGGCAGCAGCCTAGGTTAATT
ACTGTACATTTGGAACGGGGC
sequence-based reagentoGB24this paperCGCCGCCAGCCAATCTGCCCA
GCCACCTGGCTGGTGA
TCTGGGACTGTTC
sequence-based reagentoGB26this paperATGAATAAGCLCTCCGATCA
TCATATGTATATCTCCTTCTTATA
sequence-based reagentoGB27this paperGCATTTATGAAACCCGCT
GCTAATTAACCTAGGC
TGCTGCCACCG
sequence-based reagentoGB28this paperATGATCGGAGGCTTATTCATCT
sequence-based reagentoGB29this paperGCAGCGGGTTTCATAAATGCCA
sequence-based reagentoGB33this paperGGGCAGATTGGCTGGCG
GCGAGAAGGCATCAAGTA
sequence-based reagentoGB34this paperAGCAAGAGTCTGGTGCCGCG
CGGCAGCGGTAAGCAGTC
GATCGCCATTGATG
sequence-based reagentoGB35this paperCTGCTTACCGCTGCCGCG
CGGCACCAGACTCTTGCT
TGTTTCATCAGCTGTG
sequence-based reagentoGB47this paperTGAGATCCGGCTGC
TAACAAAGCC
sequence-based reagentoGB48this paperTTTAAGAAGGAGATATACA
TATGGCAGAAATCGGTACTGG
sequence-based reagentoGB49this paperAGTGCATCTCCCGTGATGC
AGAAATCTAATAAATTGCCA
sequence-based reagentoGB50this paperAGTGCATCTCCCGTGATGC
AAAACTGGACCCAGTTAGATGGC
sequence-based reagentoGB51this paperAGTGCATCTCCCGTGATGCA
GAACTGGACCCAGCCGGTGC
sequence-based reagentoGB52this paperTGCATCACGGGAGATGCACT
sequence-based reagentoGB53this paperGTTAGCAGCCGGATCTCAGT
GGTGATGATGGTGATGTTG
AAGCTGCCACAAGGCAGG
sequence-based reagentoGB174this paperAGTGCATCTCCCGTGATGC
AGCTTCCGCCGGTACCTCCAC
sequence-based reagentoGH338DOI: 10.7554/eLife.03648CATATGTATATCTCCTTCTT
ATACTTAACTAATATAC
TAAGATG
sequence-based reagentoEP642this paperCCGATATCCACGGT
TGGTGGCCCG
sequence-based reagentoEP643this paperACCAACCGTGGATATCGGG
ACTCAGAATGGCAACT
GGACCAGC
sequence-based reagentoEP644this paperTCTAGATACTTCGTCATC
CGAATTGAAGATGGA
sequence-based reagentoEP645this paperAATTCGGATGACGAAGT
ATCTAGAGTTGTCTGT
CACACTCTCCACTGCG
sequence-based reagentoGH847this paperCCAAACTGAAGGTCAAGGTGGTC
sequence-based reagentoGH848this paperCCTTGACCTTCAGTTTGGTGCGC
sequence-based reagentoGH853DOI: 10.7554/eLife.03648TAATTAACCTAGGC
TGCTGCCACCG
sequence-based reagentoGH1204this paperGTTAATTAAAACAG
ATGCACGACGGTT
sequence-based reagentoGH1205this paperGTGCATCTGTTTTAATTAACA
TGGAGGAGAGTGAGTACGAGT
sequence-based reagentoGH1206this paperGCAGCAGCCTAGGTTAATTA
GAACTGGACCCAGCCGG
sequence-based reagentoGH1227this paperGGAAGTTCTGTTCCAGG
GGCCCGGGTCCGG
CATGTCCCCT
sequence-based reagentoGH1228this paperGCCCCTGGAACAGAACTTC
CAGGCCGGATCCGCCCTTCTT
sequence-based reagentoGH1231this paperCATATGTATATCTCCTT
CTTAAAGTTAAAC
sequence-based reagentoGH1246this paperCCGCTGAGCAATAACT
AGCATAAC
sequence-based reagentoGH1247this paperCTAATGCAGGAGTCGCATAAGG
sequence-based reagentoGH1249this paperGTTATGCTAGTTAT
TGCTCAGCGG
sequence-based reagentoGH1250this paperTTATGCGACTCCTGCATTAG
GCGCGAGGCAGGATCTCG
sequence-based reagentrEP360this paperGene-specific target of crRNA:
TGAAGTGTCTCGTAACAAGA
sequence-based reagentrGB156this paperGene-specific target of crRNA:
CAAATCACGTCTCAAGTGAC
sequence-based reagentrGB155this paperGene-specific target of crRNA:
TTGGGTGAAGTTCTAGCATC
sequence-based reagentrEP254this paperGene-specific target of crRNA:
CGGGCTGTCGAGGTTCCAGT
sequence-based reagentrEP676this paperGene-specific target of crRNA:
CACAAAATATCGAGAACTAT
sequence-based reagentrEP700this paperGene-specific target of crRNA:
CCCTGGCAACGCAATTGAGG
sequence-based reagentoGB154this paperTCCCATTGGTTCGcGAAGTGTCT
CGTAACAAGATGaAAGTTAA
GGTATTTCACTTGTCAC
sequence-based reagentoGB159this paperTTCGTTACATTGGAcGATCG
GGACTGTATGAAACtAGcTGC
TAGAACTTCACCCAACCCT
sequence-based reagentoGB130this paperGGAGCAGTCACAAATCACGT
CTCAAGTtGCCGGCCAAATT
GGATGGCGTCGGGAGGGTAT
sequence-based reagentoEP674this paperTTCGCTATAAAATCCCTATTT
TTCAGAGatGCgGACTGGAAC
CTCGACAGCCCGGCTTGG
sequence-based reagentoEP680this paperCCGCCGATCGGAACCAGCGG
TCATAAAGatGCgGACTGGA
ACCTCGACAGCCCGGCTTGG
sequence-based reagentoEP701this paperGCCCGATCGATGCGCACCC
TGGCAACGCAATTGAGGCcG
TTTCgGATaacTCTaGATATTT
TGTGATTCGTTTGCAG
peptide, recombinant
protein		AcTEV ProteaseInvitrogen12575015
software, algorithmGraphPad Prism
(version 7.0 c for Mac)		GraphPad Software,
www.graphpad.com		RRID:SCR_002798
software, algorithmFijidoi:10.1038/
nmeth.2019		RRID:SCR_002285

  1. *allele generated by CRISPR

Additional files

Supplementary file 1

(A) Strains (B) Plasmids (C) Oligonucleotides.

https://doi.org/10.7554/eLife.32242.011
Transparent reporting form
https://doi.org/10.7554/eLife.32242.012

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  1. Gwendolyn M Beacham
  2. Edward A Partlow
  3. Jeffrey J Lange
  4. Gunther Hollopeter
(2018)
NECAPs are negative regulators of the AP2 clathrin adaptor complex
eLife 7:e32242.
https://doi.org/10.7554/eLife.32242