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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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Related in: MedlinePlus

An Inter-subunit salt bridge is broken in the active conformation of AP2.Predicted location of the modified worm residues within the inactive (PBD ID: 2VGL) and active (PBD ID: 2XA7) crystal structures of the vertebrate AP2 core complex. Alpha is blue, beta is green, mu2 is pink, and sigma is cyan. The residue numbers are from the worm subunits. The residues are hidden in both of these views.DOI:http://dx.doi.org/10.7554/eLife.03648.014
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fig5s1: An Inter-subunit salt bridge is broken in the active conformation of AP2.Predicted location of the modified worm residues within the inactive (PBD ID: 2VGL) and active (PBD ID: 2XA7) crystal structures of the vertebrate AP2 core complex. Alpha is blue, beta is green, mu2 is pink, and sigma is cyan. The residue numbers are from the worm subunits. The residues are hidden in both of these views.DOI:http://dx.doi.org/10.7554/eLife.03648.014

Mentions: The protease sensitivity of the suppressor mutations indicates that the closed structure determined by X-ray crystallography is an authentic structure in vivo, and that these mutations destabilize the closed state of AP2. Nevertheless, it is possible that the mapping of these mutations onto the crystal structure is coincidental. To verify that the closed structure has in vivo significance we identified a mutation among our suppressors that would disrupt a salt bridge in the closed conformation, and used the crystal structure to predict a compensatory mutation that would restore the salt bridge. In the closed conformation β (E361) forms a salt bridge to μ (K411) (Figure 2A; Figure 5A; Figure 5—figure supplement 1). We therefore analyzed mutations in β (E361K) and μ (K411E) that break this salt bridge, and found that both suppressed the fcho-1 mutant phenotype (Figure 5B). These mutations also increased protease sensitivity relative to the fcho-1 mutant, and increased phosphorylation of threonine-160 (Figure 5D,E). Similar to previous results (Figure 4), only the mutation that produced an acutely open complex (μK411E) significantly rescued the cargo-recycling defect of fcho-1 mutants (Figure 5C). We then constructed the double mutant containing both the βE361K and μK411E mutations which should restore the salt bridge. The two mutations together no longer suppressed the fcho-1 growth phenotype or cargo retrieval defect of the fcho-1 mutants, and reversed the protease sensitivity of the single mutants. Phosphorylation of T160 in the double mutant was reduced relative to the μK411E single mutant but was not fully restored to fcho(−) levels. These results confirm that the closed form as determined by crystallography predominates in the fcho-1 mutant and that destabilizing the closed form can bypass the requirement for FCHO-1.10.7554/eLife.03648.013Figure 5.Charge swaps activate and inactivate AP2 in vivo.


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)

An Inter-subunit salt bridge is broken in the active conformation of AP2.Predicted location of the modified worm residues within the inactive (PBD ID: 2VGL) and active (PBD ID: 2XA7) crystal structures of the vertebrate AP2 core complex. Alpha is blue, beta is green, mu2 is pink, and sigma is cyan. The residue numbers are from the worm subunits. The residues are hidden in both of these views.DOI:http://dx.doi.org/10.7554/eLife.03648.014
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5s1: An Inter-subunit salt bridge is broken in the active conformation of AP2.Predicted location of the modified worm residues within the inactive (PBD ID: 2VGL) and active (PBD ID: 2XA7) crystal structures of the vertebrate AP2 core complex. Alpha is blue, beta is green, mu2 is pink, and sigma is cyan. The residue numbers are from the worm subunits. The residues are hidden in both of these views.DOI:http://dx.doi.org/10.7554/eLife.03648.014
Mentions: The protease sensitivity of the suppressor mutations indicates that the closed structure determined by X-ray crystallography is an authentic structure in vivo, and that these mutations destabilize the closed state of AP2. Nevertheless, it is possible that the mapping of these mutations onto the crystal structure is coincidental. To verify that the closed structure has in vivo significance we identified a mutation among our suppressors that would disrupt a salt bridge in the closed conformation, and used the crystal structure to predict a compensatory mutation that would restore the salt bridge. In the closed conformation β (E361) forms a salt bridge to μ (K411) (Figure 2A; Figure 5A; Figure 5—figure supplement 1). We therefore analyzed mutations in β (E361K) and μ (K411E) that break this salt bridge, and found that both suppressed the fcho-1 mutant phenotype (Figure 5B). These mutations also increased protease sensitivity relative to the fcho-1 mutant, and increased phosphorylation of threonine-160 (Figure 5D,E). Similar to previous results (Figure 4), only the mutation that produced an acutely open complex (μK411E) significantly rescued the cargo-recycling defect of fcho-1 mutants (Figure 5C). We then constructed the double mutant containing both the βE361K and μK411E mutations which should restore the salt bridge. The two mutations together no longer suppressed the fcho-1 growth phenotype or cargo retrieval defect of the fcho-1 mutants, and reversed the protease sensitivity of the single mutants. Phosphorylation of T160 in the double mutant was reduced relative to the μK411E single mutant but was not fully restored to fcho(−) levels. These results confirm that the closed form as determined by crystallography predominates in the fcho-1 mutant and that destabilizing the closed form can bypass the requirement for FCHO-1.10.7554/eLife.03648.013Figure 5.Charge swaps activate and inactivate AP2 in vivo.

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