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

Hollopeter G, Lange JJ, Zhang Y, Vu TN, Gu M, Ailion M, Lambie EJ, Slaughter BD, Unruh JR, Florens L, Jorgensen EM - Elife (2014)

Bottom Line: Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation.We screened for Caenorhabditis elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants.The domain of FCHo that induces this rearrangement is not the F-BAR domain or the µ-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, United States.

ABSTRACT
The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for Caenorhabditis elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 s identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the µ-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2.

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Suppression of fcho-1 by missense mutations in individual AP2 subunits.For (A–C), the AP2 mutation identified in each suppressed fcho-1 mutant strain is indicated. Only a subset of the mutations were assayed. See Figure 2—figure supplement 2 for complete list of suppressor mutations. All data represent the mean ± SEM. (A) Starvation assay (days required for a worm population to expand and consume the bacterial food). The AP2; fcho-1 double mutants all exhibit faster starvation rates compared to fcho-1 mutants alone (p < 0.01, unpaired, two-tailed t-test n ≥ 9). (B) Cargo assay. Fluorescence from GFP-tagged cargo on plasma membrane of intestinal cells. Left and right panels indicate experiments conducted on different days. n ≥ 5; *p < 0.05 and **p < 0.01, unpaired, two-tailed t-test compared to fcho(−) alone; † data reported in Figure 1E caption. (C) FRAP assay. Average time constants for fluorescence recovery after photobleaching of GFP-tagged AP2. n ≥ 3; *p < 0.05, unpaired, two-tailed t-test compared to fcho(−) alone.DOI:http://dx.doi.org/10.7554/eLife.03648.007
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fig2s1: Suppression of fcho-1 by missense mutations in individual AP2 subunits.For (A–C), the AP2 mutation identified in each suppressed fcho-1 mutant strain is indicated. Only a subset of the mutations were assayed. See Figure 2—figure supplement 2 for complete list of suppressor mutations. All data represent the mean ± SEM. (A) Starvation assay (days required for a worm population to expand and consume the bacterial food). The AP2; fcho-1 double mutants all exhibit faster starvation rates compared to fcho-1 mutants alone (p < 0.01, unpaired, two-tailed t-test n ≥ 9). (B) Cargo assay. Fluorescence from GFP-tagged cargo on plasma membrane of intestinal cells. Left and right panels indicate experiments conducted on different days. n ≥ 5; *p < 0.05 and **p < 0.01, unpaired, two-tailed t-test compared to fcho(−) alone; † data reported in Figure 1E caption. (C) FRAP assay. Average time constants for fluorescence recovery after photobleaching of GFP-tagged AP2. n ≥ 3; *p < 0.05, unpaired, two-tailed t-test compared to fcho(−) alone.DOI:http://dx.doi.org/10.7554/eLife.03648.007

Mentions: To identify components downstream of FCHo, we performed a genetic screen for mutations that suppress a mutation in fcho-1. To increase the probability of getting missense mutations we used the mutagen N-ethyl-N-nitrosourea (ENU), which can generate transversions and can therefore swap charges, or hydrophilic and hydrophobic amino acids. In addition, we designed a multigenerational screen to select for subtle improvements in fitness. Wild-type animals grow rapidly and starve a culture plate in 5 days, whereas fcho-1 mutants exhibit reduced fecundity (Figure1—figure supplement 1E) and require twice as long to consume the same amount of food (Figure 1—figure supplement 1F). We selected for suppressors that rapidly starved plates, and identified 71 dominant mutations that confer increased fitness to fcho-1 mutants and suppressed the jowls phenotype. All of these suppressed strains contained second site missense mutations in one of the four subunits of AP2 (Figure 2—figure supplement 2) and none exhibited loss-of-function phenotypes for these adaptin genes (Figure 2—figure supplement 1A–C). These mutations all occur at conserved amino acids, and cluster at sites likely to stabilize the closed (inactive) conformation when placed on the crystal structures of AP2 (Figure 2A–C) (Collins et al., 2002; Kelly et al., 2008; Jackson et al., 2010). These mutations can be classified into four groups: (1) residues that lie in the bowl-like interface between the mu2 subunit and the other three subunits, (2) residues that stabilize the insertion of the N-terminus of the beta subunit into the cargo binding motif of sigma, (3) residues in the alpha subunit that are found in the helical solenoid that lies across the top of the complex, and (4) the phosphorylation site on the mu2 subunit. It is likely that these mutations destabilize the closed conformation of AP2, suggesting that the open conformation of AP2 may bypass the requirement for FCHo. In other words, these mutations would promote an open conformation of AP2, suggesting that AP2 may dwell in the closed state in the absence of FCHo.10.7554/eLife.03648.006Figure 2.Mutations in AP2 closed conformation interfaces suppress fcho(−).


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

Hollopeter G, Lange JJ, Zhang Y, Vu TN, Gu M, Ailion M, Lambie EJ, Slaughter BD, Unruh JR, Florens L, Jorgensen EM - Elife (2014)

Suppression of fcho-1 by missense mutations in individual AP2 subunits.For (A–C), the AP2 mutation identified in each suppressed fcho-1 mutant strain is indicated. Only a subset of the mutations were assayed. See Figure 2—figure supplement 2 for complete list of suppressor mutations. All data represent the mean ± SEM. (A) Starvation assay (days required for a worm population to expand and consume the bacterial food). The AP2; fcho-1 double mutants all exhibit faster starvation rates compared to fcho-1 mutants alone (p < 0.01, unpaired, two-tailed t-test n ≥ 9). (B) Cargo assay. Fluorescence from GFP-tagged cargo on plasma membrane of intestinal cells. Left and right panels indicate experiments conducted on different days. n ≥ 5; *p < 0.05 and **p < 0.01, unpaired, two-tailed t-test compared to fcho(−) alone; † data reported in Figure 1E caption. (C) FRAP assay. Average time constants for fluorescence recovery after photobleaching of GFP-tagged AP2. n ≥ 3; *p < 0.05, unpaired, two-tailed t-test compared to fcho(−) alone.DOI:http://dx.doi.org/10.7554/eLife.03648.007
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2s1: Suppression of fcho-1 by missense mutations in individual AP2 subunits.For (A–C), the AP2 mutation identified in each suppressed fcho-1 mutant strain is indicated. Only a subset of the mutations were assayed. See Figure 2—figure supplement 2 for complete list of suppressor mutations. All data represent the mean ± SEM. (A) Starvation assay (days required for a worm population to expand and consume the bacterial food). The AP2; fcho-1 double mutants all exhibit faster starvation rates compared to fcho-1 mutants alone (p < 0.01, unpaired, two-tailed t-test n ≥ 9). (B) Cargo assay. Fluorescence from GFP-tagged cargo on plasma membrane of intestinal cells. Left and right panels indicate experiments conducted on different days. n ≥ 5; *p < 0.05 and **p < 0.01, unpaired, two-tailed t-test compared to fcho(−) alone; † data reported in Figure 1E caption. (C) FRAP assay. Average time constants for fluorescence recovery after photobleaching of GFP-tagged AP2. n ≥ 3; *p < 0.05, unpaired, two-tailed t-test compared to fcho(−) alone.DOI:http://dx.doi.org/10.7554/eLife.03648.007
Mentions: To identify components downstream of FCHo, we performed a genetic screen for mutations that suppress a mutation in fcho-1. To increase the probability of getting missense mutations we used the mutagen N-ethyl-N-nitrosourea (ENU), which can generate transversions and can therefore swap charges, or hydrophilic and hydrophobic amino acids. In addition, we designed a multigenerational screen to select for subtle improvements in fitness. Wild-type animals grow rapidly and starve a culture plate in 5 days, whereas fcho-1 mutants exhibit reduced fecundity (Figure1—figure supplement 1E) and require twice as long to consume the same amount of food (Figure 1—figure supplement 1F). We selected for suppressors that rapidly starved plates, and identified 71 dominant mutations that confer increased fitness to fcho-1 mutants and suppressed the jowls phenotype. All of these suppressed strains contained second site missense mutations in one of the four subunits of AP2 (Figure 2—figure supplement 2) and none exhibited loss-of-function phenotypes for these adaptin genes (Figure 2—figure supplement 1A–C). These mutations all occur at conserved amino acids, and cluster at sites likely to stabilize the closed (inactive) conformation when placed on the crystal structures of AP2 (Figure 2A–C) (Collins et al., 2002; Kelly et al., 2008; Jackson et al., 2010). These mutations can be classified into four groups: (1) residues that lie in the bowl-like interface between the mu2 subunit and the other three subunits, (2) residues that stabilize the insertion of the N-terminus of the beta subunit into the cargo binding motif of sigma, (3) residues in the alpha subunit that are found in the helical solenoid that lies across the top of the complex, and (4) the phosphorylation site on the mu2 subunit. It is likely that these mutations destabilize the closed conformation of AP2, suggesting that the open conformation of AP2 may bypass the requirement for FCHo. In other words, these mutations would promote an open conformation of AP2, suggesting that AP2 may dwell in the closed state in the absence of FCHo.10.7554/eLife.03648.006Figure 2.Mutations in AP2 closed conformation interfaces suppress fcho(−).

Bottom Line: Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation.We screened for Caenorhabditis elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants.The domain of FCHo that induces this rearrangement is not the F-BAR domain or the µ-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, United States.

ABSTRACT
The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for Caenorhabditis elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 s identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the µ-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2.

Show MeSH
Related in: MedlinePlus