Edward A Partlow, Kevin S Cannon, Gunther Hollopeter*, Richard W Baker*. Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor complex for cargo internalization (2022). Nat. Struct. Mol. Biol. *co-corresponding authors.
Gwendolyn M Beacham, Derek T Wei*, Erika Beyrent*, Ying Zhang, Jian Zheng, Mari MK Camacho, Laurence Florens, Gunther Hollopeter. The C. elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator (submitted 2/18/2022). *contributed equally.
Gwendolyn M Beacham, Edward A Partlow, Gunther Hollopeter. Conformational regulation of AP1 and AP2 clathrin adaptor complexes (2019). Traffic 10:741-751.
Edward A Partlow, Richard W Baker*, Gwendolyn M Beacham, Joshua S Chappie, Andres E Leschziner*, Gunther Hollopeter*. A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex (2019). eLife 8:e50003. *co-corresponding authors.
first posted to bioRxiv on July 13, 2019
Relevance: This study is a joint effort between Ed Partlow and the structural biology superstar @therickybaker from the lab of @aleschziner. The study follows up on discoveries reported in @gwen_beacham ‘s paper https://elifesciences.org/articles/32242.
AP2 is the central organizing hub of clathrin-mediated endocytosis. A phosphorylation event on AP2 coincides with conformational rearrangement to the open, active state but the functional significance of this modification is unclear, and it is missing in all structures.
We know almost nothing about how AP2 is inactivated once the complex achieves the active state, even though imaging data suggests endocytic pits often abort, and adaptors are clearly recycled after endocytosis. We reveal a potential mechanism for regulated inactivation of AP2.
We previously found that loss of NECAP restores AP2 activity in C. elegans lacking the AP2 activator, FCHo. This suggested NECAP counterbalances AP2 activation. Without NECAP, AP2 accumulated in an open and phosphorylated state, and NECAP bound open and phosphorylated AP2.
Based on these results, we proposed that NECAP must somehow recognize active AP2 complexes and facilitate their return to the inactive state (closed and dephosphorylated). However, we lacked a mechanism and evidence that NECAP directly inactivated AP2.
Reviewers cleverly suggested we characterize two missense mutations isolated in the PHear domain of NECAP. Because they disrupted binding to phosphorylated AP2, but not to open unphosphorylated AP2, we wondered if open and phosphorylated forms of AP2 are distinguished by NECAP.
To understand the mechanism of NECAP action we designed a genetic screen to disrupt the NECAP-AP2 interface, developed methods to control and query to conformation of AP2 in vitro, and collaborated with @therickybaker and @aleschziner to visualize complexes using cryo-EM.
We observe the NECAP PHear domain directly bound to the AP2 phosphorylation mark, explaining why we isolate threonine mutations in worms. Binding to the phosphorylated threonine is also coordinated by residues of the PHear domain that were isolated our genetic screens.
To stimulate opening of AP2 in vitro, we use DNA oligos and heparin act as membrane mimetics. This simple technique enables us to discover that another domain of NECAP specifically recognizes open AP2 and acting together, the two domains cooperate to directly clamp AP2 closed.
Gwendolyn M Beacham, Edward A Partlow, Jeffrey J Lange, Gunther Hollopeter. NECAPs are negative regulators of the clathrin adaptor complex (2018). eLife 10.7554/eLife.32242.
first posted to bioRxiv on September 22, 2017
Relevance: This work describes our discovery of a novel mechanism for counterbalancing AP2 activity at the appropriate time and place. Using genetic analysis in C. elegans, we found that AP2 activity can also be restored in fcho-1 mutants (see publication below) by inactivating ncap-1, the sole gene encoding an adaptin-ear-binding coat-associated protein (NECAP). This class of proteins is highly conserved from humans to plants but has remained functionally enigmatic. Using biochemical assays, we observed that AP2 accumulates in an open and phosphorylated state in ncap-1 mutants. These results imply that NECAPs normally inactivate AP2. Indeed, we showed that NECAPs preferentially bind active forms of AP2 in vitro and localize with constitutively active AP2 in vivo. We propose that NECAPs primarily function late in endocytosis to negatively regulate AP2.
SELECTED PUBLICATIONS FROM GUNTHER'S TRAINING:
Hollopeter G, Lange JJ, Zhang Y, Vu TV, Gu M, Ailion M, Lambie EJ, Slaughter BD, Unruh JR, Florens L, Jorgensen EM. The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex (2014). eLife 10.7554/eLife.03648.
Relevance: Clathrin-mediated endocytosis is the predominant method eukaryotic cells use to engulf membrane proteins, lipids and extracellular material. The AP2 clathrin adaptor complex is the molecular keystone of this process, but it has been difficult to determine how AP2 initiates endocytosis. Using large-scale genetic screens in combination with new biochemical and imaging assays in C. elegans, we discovered that a conserved class of membrane-associated proteins promotes a conformational rearrangement of AP2 to launch endocytosis.
Liu Q, Hollopeter G, Jorgensen EM. Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction (2009). Proc. Natl. Acad. Sci. 106:10823-8.
Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D, Conley PB. Identification of the platelet ADP receptor targeted by antithrombotic drugs (2001). Nature 409: 202-207.
Erickson JC, Hollopeter G, Palmiter RD. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y (1996). Science 274:1704-1707.
Gwendolyn M Beacham, Derek T Wei*, Erika Beyrent*, Ying Zhang, Jian Zheng, Mari MK Camacho, Laurence Florens, Gunther Hollopeter. The C. elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator (submitted 2/18/2022). *contributed equally.
Gwendolyn M Beacham, Edward A Partlow, Gunther Hollopeter. Conformational regulation of AP1 and AP2 clathrin adaptor complexes (2019). Traffic 10:741-751.
Edward A Partlow, Richard W Baker*, Gwendolyn M Beacham, Joshua S Chappie, Andres E Leschziner*, Gunther Hollopeter*. A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex (2019). eLife 8:e50003. *co-corresponding authors.
first posted to bioRxiv on July 13, 2019
Relevance: This study is a joint effort between Ed Partlow and the structural biology superstar @therickybaker from the lab of @aleschziner. The study follows up on discoveries reported in @gwen_beacham ‘s paper https://elifesciences.org/articles/32242.
AP2 is the central organizing hub of clathrin-mediated endocytosis. A phosphorylation event on AP2 coincides with conformational rearrangement to the open, active state but the functional significance of this modification is unclear, and it is missing in all structures.
We know almost nothing about how AP2 is inactivated once the complex achieves the active state, even though imaging data suggests endocytic pits often abort, and adaptors are clearly recycled after endocytosis. We reveal a potential mechanism for regulated inactivation of AP2.
We previously found that loss of NECAP restores AP2 activity in C. elegans lacking the AP2 activator, FCHo. This suggested NECAP counterbalances AP2 activation. Without NECAP, AP2 accumulated in an open and phosphorylated state, and NECAP bound open and phosphorylated AP2.
Based on these results, we proposed that NECAP must somehow recognize active AP2 complexes and facilitate their return to the inactive state (closed and dephosphorylated). However, we lacked a mechanism and evidence that NECAP directly inactivated AP2.
Reviewers cleverly suggested we characterize two missense mutations isolated in the PHear domain of NECAP. Because they disrupted binding to phosphorylated AP2, but not to open unphosphorylated AP2, we wondered if open and phosphorylated forms of AP2 are distinguished by NECAP.
To understand the mechanism of NECAP action we designed a genetic screen to disrupt the NECAP-AP2 interface, developed methods to control and query to conformation of AP2 in vitro, and collaborated with @therickybaker and @aleschziner to visualize complexes using cryo-EM.
We observe the NECAP PHear domain directly bound to the AP2 phosphorylation mark, explaining why we isolate threonine mutations in worms. Binding to the phosphorylated threonine is also coordinated by residues of the PHear domain that were isolated our genetic screens.
To stimulate opening of AP2 in vitro, we use DNA oligos and heparin act as membrane mimetics. This simple technique enables us to discover that another domain of NECAP specifically recognizes open AP2 and acting together, the two domains cooperate to directly clamp AP2 closed.
Gwendolyn M Beacham, Edward A Partlow, Jeffrey J Lange, Gunther Hollopeter. NECAPs are negative regulators of the clathrin adaptor complex (2018). eLife 10.7554/eLife.32242.
first posted to bioRxiv on September 22, 2017
Relevance: This work describes our discovery of a novel mechanism for counterbalancing AP2 activity at the appropriate time and place. Using genetic analysis in C. elegans, we found that AP2 activity can also be restored in fcho-1 mutants (see publication below) by inactivating ncap-1, the sole gene encoding an adaptin-ear-binding coat-associated protein (NECAP). This class of proteins is highly conserved from humans to plants but has remained functionally enigmatic. Using biochemical assays, we observed that AP2 accumulates in an open and phosphorylated state in ncap-1 mutants. These results imply that NECAPs normally inactivate AP2. Indeed, we showed that NECAPs preferentially bind active forms of AP2 in vitro and localize with constitutively active AP2 in vivo. We propose that NECAPs primarily function late in endocytosis to negatively regulate AP2.
SELECTED PUBLICATIONS FROM GUNTHER'S TRAINING:
Hollopeter G, Lange JJ, Zhang Y, Vu TV, Gu M, Ailion M, Lambie EJ, Slaughter BD, Unruh JR, Florens L, Jorgensen EM. The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex (2014). eLife 10.7554/eLife.03648.
Relevance: Clathrin-mediated endocytosis is the predominant method eukaryotic cells use to engulf membrane proteins, lipids and extracellular material. The AP2 clathrin adaptor complex is the molecular keystone of this process, but it has been difficult to determine how AP2 initiates endocytosis. Using large-scale genetic screens in combination with new biochemical and imaging assays in C. elegans, we discovered that a conserved class of membrane-associated proteins promotes a conformational rearrangement of AP2 to launch endocytosis.
Liu Q, Hollopeter G, Jorgensen EM. Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction (2009). Proc. Natl. Acad. Sci. 106:10823-8.
Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D, Conley PB. Identification of the platelet ADP receptor targeted by antithrombotic drugs (2001). Nature 409: 202-207.
Erickson JC, Hollopeter G, Palmiter RD. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y (1996). Science 274:1704-1707.