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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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Recessive alleles isolated from genetic screen for ‘jowls’ phenotype * independently identified ‘jowls’ mutant.DOI:http://dx.doi.org/10.7554/eLife.03648.005
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fig1s2: Recessive alleles isolated from genetic screen for ‘jowls’ phenotype * independently identified ‘jowls’ mutant.DOI:http://dx.doi.org/10.7554/eLife.03648.005

Mentions: Mutations in the AP2 complex alpha and mu subunits in C. elegans (encoded by the apa-2 and apm-2 genes) result in animals with pleiotropic phenotypes including reduced body length (Dpy), egg-laying defects (Egl) and uncoordinated locomotion (Unc). In addition, they exhibit a unique ‘jowls’ phenotype, in which the mutants exhibit bulges in the cuticle on either side of the head (Gu et al., 2013). Deletion of the sigma subunit (aps-2) produces a similar ‘jowls’ phenotype (Figure 1A and Figure 1—figure supplement 1B), while the beta subunit is shared by both AP1 and AP2 in C. elegans and mutations in apb-1 are lethal. We screened for mutants with the jowls phenotype and identified multiple mutations in three genes coding for alpha adaptin, mu2 adaptin and the nematode homolog of FCHo (Figure 1—figure supplement 2). We generated a deletion allele fcho-1(ox477) by transposon excision (Figure 1—figure supplement 1A); all six mutant alleles of fcho-1 produced defects strikingly similar to mutants lacking AP2 subunits, including the ‘jowls’ phenotype (Figure 1A and Figure 1—figure supplement 1B), suggesting that AP2 function is compromised in the absence of FCHo.10.7554/eLife.03648.003Figure 1.Loss of FCHo compromises AP2 activity.


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)

Recessive alleles isolated from genetic screen for ‘jowls’ phenotype * independently identified ‘jowls’ mutant.DOI:http://dx.doi.org/10.7554/eLife.03648.005
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215536&req=5

fig1s2: Recessive alleles isolated from genetic screen for ‘jowls’ phenotype * independently identified ‘jowls’ mutant.DOI:http://dx.doi.org/10.7554/eLife.03648.005
Mentions: Mutations in the AP2 complex alpha and mu subunits in C. elegans (encoded by the apa-2 and apm-2 genes) result in animals with pleiotropic phenotypes including reduced body length (Dpy), egg-laying defects (Egl) and uncoordinated locomotion (Unc). In addition, they exhibit a unique ‘jowls’ phenotype, in which the mutants exhibit bulges in the cuticle on either side of the head (Gu et al., 2013). Deletion of the sigma subunit (aps-2) produces a similar ‘jowls’ phenotype (Figure 1A and Figure 1—figure supplement 1B), while the beta subunit is shared by both AP1 and AP2 in C. elegans and mutations in apb-1 are lethal. We screened for mutants with the jowls phenotype and identified multiple mutations in three genes coding for alpha adaptin, mu2 adaptin and the nematode homolog of FCHo (Figure 1—figure supplement 2). We generated a deletion allele fcho-1(ox477) by transposon excision (Figure 1—figure supplement 1A); all six mutant alleles of fcho-1 produced defects strikingly similar to mutants lacking AP2 subunits, including the ‘jowls’ phenotype (Figure 1A and Figure 1—figure supplement 1B), suggesting that AP2 function is compromised in the absence of FCHo.10.7554/eLife.03648.003Figure 1.Loss of FCHo compromises AP2 activity.

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