Structural and functional characterization of cargo-binding sites on the μ4-subunit of adaptor protein complex 4

Breyan H. Ross, Yimo Lin, Esteban A. Corales, Patricia V. Burgos, Gonzalo A. Mardones

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25 Scopus citations

Abstract

Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in m subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the m4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of m4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of m4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the noncanonical YXXØ-signal of APP is limited to the non-canonical site of μ4.

Original languageEnglish
Article numbere88147
JournalPLoS ONE
Volume9
Issue number2
DOIs
StatePublished - 2014
Externally publishedYes

Bibliographical note

Funding Information:
We thank X. Zhu, and N. Tsai for excellent technical assistance; J. Bonifacino and R. Mattera for kindly providing HA-epitope-tagged μ4 constructs, and the use of the iTC200 microcalorimeter; J. Hurley for lab space and the use of the Mosquito liquid handler; Daniel Kloer for help with crystallographic data analysis; H. Richter and C. Spichiger for the use of the Rotor-Gene Q real-time rotary analyzer; X. Ren for help with N-terminal sequencing; R. Hegde for the anti-HA-epitope serum; J. Hirst and M. Robinson for antibodies to AP-4 subunits; V. Cavieres for help with immunoblotting; Peter McCormick and Thomas Wollert for critically reading the manuscript; and the staff of the SER-CAT beamline at the APS, Argonne National Laboratory, for assistance with X-ray data collection. Use of the APS was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract No. DE-AC02-06CH11357.

ASJC Scopus subject areas

  • General

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