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Binding of Cargo Sorting Signals to AP-1 Enhances Its Association with ADP Ribosylation Factor 1-GTP Intaek Lee Washington University School of Medicine in St

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Binding of Cargo Sorting Signals to AP-1 Enhances Its Association with ADP Ribosylation Factor 1-GTP Intaek Lee Washington University School of Medicine in St Washington University School of Medicine Digital Commons@Becker Open Access Publications 2-4-2008 Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1-GTP Intaek Lee Washington University School of Medicine in St. Louis Balraj Doray Washington University School of Medicine in St. Louis Jennifer Govero Washington University School of Medicine in St. Louis Stuart Kornfeld Washington University School of Medicine in St. Louis Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Part of the Medicine and Health Sciences Commons Recommended Citation Lee, Intaek; Doray, Balraj; Govero, Jennifer; and Kornfeld, Stuart, ,"Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1-GTP." The ourJ nal of Cell Biology.,. 467-472. (2008). https://digitalcommons.wustl.edu/open_access_pubs/565 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Published February 4, 2008 JCB: REPORT Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1 – GTP Intaek Lee , Balraj Doray , Jennifer Govero , and Stuart Kornfeld Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110 he adaptor protein AP-1 is the major coat protein we provide evidence for cross talk between the dileu- involved in the formation of clathrin-coated vesicles cine and tyrosine binding sites within the AP-1 core T at the trans-Golgi network. The prevailing view is domain such that binding of a cargo signal to one site fa- that AP-1 recruitment involves coincident binding to multi- cilitates binding to the other site. The stable association of ple low-affi nity sites comprising adenosine diphosphate AP-1 with Arf-1 – GTP, which is induced by cargo signals, ribosylation factor 1 (Arf-1)– guanosine triphosphate (GTP), would serve to provide suffi cient time for adaptor polym- Downloaded from cargo sorting signals, and phosphoinositides. We now erization and clathrin recruitment while ensuring the show that binding of cargo signal peptides to AP-1 in- packaging of cargo molecules into the forming trans- duces a conformational change in its core domain that port vesicles. greatly enhances its interaction with Arf-1– GTP. In addition, jcb.rupress.org Introduction The adaptor protein complex AP-1 (a heterotetramer composed of AP-1. This concept of coincidence detection suggests that the of ␥ , ␤ 1, ␮ 1, and ␴ 1 subunits) plays a major role in the assem- association of AP-1 with the various TGN components, though bly of clathrin-coated vesicles (CCVs) at the TGN, serving to insuffi cient in themselves, is signifi cantly enhanced through on November 8, 2011 select and link cargo molecules with the growing clathrin lattice. multiple simultaneous interactions that together result in the AP-1 binds cargo molecules, mainly via two types of sorting proper membrane targeting of AP-1 ( Carlton and Cullen, 2005 ). determinants, a tyrosine-based YXX␾ motif (where ␾ is a bulky In support of this hypothesis, it has been demonstrated that a hydrophobic residue), which binds to the ␮ 1 subunit, and the membrane-anchored tyrosine-based sorting signal, together with dileucine-based [D/E]XXXL[L/I/M] sequence, which binds to the myristoylated Arf-1 – GTP, constitutes a minimal machinery for ␥ / ␴ 1 hemicomplex ( Traub, 2005 ). The prevailing model of AP-1 the recruitment of AP-1 to chemically defined liposomes and targeting to the TGN ascribes a major role to the small GTPase that the process can be further stimulated by specifi c phosphoino- ADP ribosylation factor 1 (Arf-1) as the primary docking site sitides ( Crottet et al., 2002 ; Baust et al., 2006 ). This fi nding that THE JOURNAL OF CELL BIOLOGY in the initial recruitment step. Activation of Arf-1 involves GTP- myristoylated Arf-1 alone cannot recruit AP-1 to liposomes is for-GDP exchange, which is catalyzed by guanine nucleotide ex- indicative of the fact that whatever conformational switch occurs change factors, which in turn exposes the covalently linked in the membrane-associated Arf-1 is insuffi cient for binding AP-1. myristoyl moiety on the Arf-1 allowing it to insert into mem- An alternate mechanism, which is not mutually exclusive branes. It is thought that the ensuing conformational change in the to the coincident detection hypothesis, is one where sorting sig- membrane-associated GTP-bound form of Arf-1 is what allows nal binding to AP-1 infl uences its association with Arf-1 – GTP. it to weakly associate with and initially recruit AP-1 ( Edeling et al., To distinguish between these two models, we synthesized soluble 2006 ). Additional components of the TGN, such as phospho ino- peptides corresponding to known dileucine- or tyrosine-based sitides and the cytosolic domains of sorting signal – bearing cargo sorting signals and determined their effects on the interaction proteins, are believed to function together with Arf-1 in providing of AP-1 with activated Arf-1. Our experiments show that both a combinatorial targeting mechanism for the Golgi localization types of sorting determinants are able to strongly enhance the AP-1 – Arf-1 – GTP interaction. We further demonstrate that the I. Lee and B. Doray contributed equally to this paper. peptide-induced stimulation of this interaction is accompanied Correspondence to Stuart Kornfeld: [email protected] by a conformational change in the adaptor core domain, which Abbreviations used in this paper: Arf-1, ADP ribosylation factor 1; BAC, bovine adrenal cytosol; BBC, bovine brain cytosol; CCV, clathrin-coated vesicle; we propose serves to stabilize the association of AP-1 with CI-MPR, cation-independent mannose 6-phosphate receptor; WT, wild-type. Golgi membranes. © The Rockefeller University Press $30.00 The Journal of Cell Biology, Vol. 180, No. 3, February 11, 2008 467–472 http://www.jcb.org/cgi/doi/10.1083/jcb.200709037 JCB 467 Published February 4, 2008 Downloaded from jcb.rupress.org on November 8, 2011 Figure 1. Binding of the CI-MPR ETEWLM peptide to AP-1 stimulates its association with activated Arf-1. (A) Recruitment of CCV-derived AP-1 to soybean liposomes is greatly enhanced only in the presence of 250 μ M ETEWLM peptide (top, compare lanes 5 and 6) even though similar amounts of activated Arf-1 are found on the liposomes, as seen in the Ponceau stain of the blot (bottom, *). Lane 1 represents 5% of the CCV coat fraction input. (B) Recruitment of 25 nM AP-1 purifi ed from BAC to liposomes is stimulated by the ETEWLM peptide (lanes 5 vs. 7) in a GTP␥ S-dependent manner (lanes 3 vs. 5). The reactions contained 4 μ M of myristoylated Arf-1, 100 μ M GTP ␥ S, and the indicated concentrations of AP-1. Lane 1 represents 20% of the input from the reaction containing 25 nM AP-1. (C) Pulldown of AP-1 from BBC with both WT GST – Arf-1 and the Q71L mutant preloaded with 2mM GTP or 100 μ M GTP ␥ S is dependent on the presence of the ETEWLM peptide. No binding to AP-2 was detected under these conditions. (D and E) Binding of AP-1, purifi ed from BAC, to GST – Arf-1 is a function of the concentration of the ETEWLM peptide (D) and purifi ed adaptor (E). The AP-1 concentration was kept constant at 25 nM in D, whereas the peptide concentrations were kept constant at 250 μ M in E. The doublet observed on the blots in D and E, and in subsequent experiments where BAC served as the source of cytosolic AP-1, is the consequence of partial proteolysis of the appendage domain of the ␥ subunit of AP-1 in our adrenal cytosol preparations. The ␥ subunit of AP-1 in our bovine brain CCV coat preparations did not undergo such proteolysis, as shown in A. (F) The dileucine peptide – dependent binding of AP-1 to activated GST – Arf-1 requires the tetramer because neither hemicomplex bound. (compare lanes 5 and 6). This recruitment required activated Arf-1, Results and discussion as no AP-1 association occurred in the absence of myristoylated AP-1 binding to Arf-1– GTP is stimulated by Arf-1 – GTP ␥ S ( Fig. 1 A , lane 4). To rule out the idea that some dileucine- and tyrosine-based sorting signals other component of the CCV coat fraction mediated the peptide- In considering a possible role for sorting signals in the modula- stimulated association of AP-1 with liposome, recruitment assays tion of AP-1 function at the TGN, we fi rst tested whether a soluble were performed with purifi ed AP-1. As in Fig. 1 A , the WT 14-aa peptide molecule corresponding to the cation-independent ETEWLM ( Fig. 1 B , lanes 4 and 5), but not the mutant ATEWAA, mannose 6-phosphate receptor (CI-MPR) internal dileucine- peptide ( Fig. 1 B , lanes 6 and 7) stimulated recruitment of puri- type sequence (ETEWLM) could affect AP-1 binding to acti- fi ed AP-1 in a GTP ␥ S-dependent manner. The ETEWLM peptide vated Arf-1 in a liposome recruitment assay. As shown in Fig. 1 A , also stimulated binding of cytosolic AP-1 to Arf-1, immobilized recruitment of AP-1 from a CCV coat fraction was greatly in- as a GST fusion protein. Fig. 1 C shows that AP-1 from bovine creased in the presence of the wild-type (WT) ETEWLM peptide brain cytosol (BBC) failed to bind either WT GST –Arf-1 or the 468 JCB • VOLUME 180 • NUMBER 3 • 2008 Published February 4, 2008 Figure 2.
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