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. 2016 Apr;17(4):400-15.
doi: 10.1111/tra.12375. Epub 2016 Mar 4.

Molecular Basis for the Interaction Between AP4 β4 and its Accessory Protein, Tepsin

Meredith N Frazier  1   2 , Alexandra K Davies  3 , Markus Voehler  2   4 , Amy K Kendall  1   2 , Georg H H Borner  5 , Walter J Chazin  2   4 , Margaret S Robinson  3 , Lauren P Jackson  1   2
Affiliations

Molecular Basis for the Interaction Between AP4 β4 and its Accessory Protein, Tepsin

Meredith N Frazier et al. Traffic. 2016 Apr.

Abstract

The adaptor protein 4 (AP4) complex (ϵ/β4/μ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 β4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal β4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the β4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on β4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function.

Keywords: adaptor protein complexes; biochemistry; cell biology; membrane trafficking; non-clathrin coats; structural biology; vesicle coats.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. The AP4 β4 appendage domain interacts directly with the tepsin C-terminus
A) Schematics of AP4 coat protein complex (top, ε/β4/μ4/σ4 subunits) and tepsin (bottom). Tepsin contains structured ENTH and VHS-like domains at its N-terminus, together with a mostly unstructured C-terminus. The conserved hydrophobic sequence for binding to β4 identified in this work (LFxG[M/L]x[L/V]) is highlighted. B) Pulldown experiments using recombinant purified proteins identified a region in the tepsin C-terminus that binds β4; a panel of GST-tepsin constructs was used as bait and β4 appendage domain as prey. (Top: Coomassie-stained SDS- PAGE gel with tepsin construct amino acid residue ranges marked; bottom: Western blot using anti-β4 antibody).
Figure 2
Figure 2. Mutagenesis of a conserved hydrophobic sequence in tepsin reduces or abolishes binding to β4
A) Sequence alignment of tepsin from major eukaryotic super groups reveals a conserved stretch of 8 residues in the unstructured C-terminus. Asterisks mark highly conserved amino acids. B) Wild-type β4 binds a recombinant tepsin fragment (residues 450–500) containing the conserved sequence with a KD of 2.9±0.8 μM by isothermal titration calorimetry (ITC, 10 independent experiments). In contrast, the tepsin fragment cannot bind purified β1 or β2 appendages found in the clathrin adaptors AP1 or AP2, respectively. C) Representative ITC traces of tepsin mutants with WT β4. Tepsin residues marked by asterisks in A) were mutated to test their importance for binding. All mutants either reduced or abrogated measurable binding to β4 in vitro. D) ITC summary results table. All KD values are represented as averages ± standard deviation; nb= no measurable binding.
Figure 3
Figure 3. NMR chemical shift perturbations reveal residues on β4 surface involved in tepsin binding
A) 15N-1H HSQC spectra of the initial (black) and final (red) titration points show chemical shifts resulting from binding upon addition of unlabeled recombinant tepsin (residues 450– 500) to labeled β4. Residues identified for further analysis are highlighted below. B) From the HSQC titration, residues that exhibited large chemical shift perturbations were mapped onto the structure (PDB 2MJ7): E632, W635, L636, I669, A670, Y682. C) Close-up view of binding interface residues identified by chemical shift perturbations.
Figure 4
Figure 4. Structure-based mutagenesis of key β4 residues eliminates in vitro interaction with tepsin
A) The β4 mutant I669A/A670S exhibits no measurable binding to the tepsin motif by ITC (representative trace). B) Wild-type GST-tepsin (residues 450–500) pulls down wild-type β4 (positive control) but fails to pull down the β4 mutant Y682V. GST (lane 4) was used as a negative control. C) Table summarizing β4 mutant results from ITC and pulldown experiments.
Figure 5
Figure 5. Tepsin is not recruited to the membrane in AP4 β4 knockout (KO) HeLa cells generated using CRISPR technology
A) Western blots of whole cell lysates from wild-type and AP4 β KO HeLa cells, probed with antibodies against AP4 β, ε, and tepsin (n.b. tepsin has two isoforms). AP4 β (marked by the arrowhead) is undetectable in the AP4 β KO cells. The band marked with the asterisk is non-specific. While the amount of AP4 ε in the AP4 β KO cells is reduced, the expression level of tepsin is unchanged. An antibody against clathrin was used as a loading control. B) Immunofluorescence double labeling for AP4 ε and tepsin in wild-type and AP4 β KO HeLa cells. In wild-type cells, tepsin labeling is punctate and concentrated in the perinuclear region where it colocalizes extensively with AP4. This pattern was absent in the AP4 β KO cells. Scale bars are 20 µm.
Figure 6
Figure 6. Disruption of the β4 appendage-tepsin interaction in vivo greatly reduces, but does not abolish, tepsin binding to AP4
A) Western blots of immunoprecipitates of AP4 β or ε from extracts of control (wild-type HeLa), AP4 β KO, or AP4 β KO cells stably rescued with full-length wild-type or mutant (earless, Y682V, or I669A/A670S) β4, probed with antibodies against AP4 β, ε, and tepsin (marked by arrow heads). No tepsin could be detected in the immunoprecipitates from the AP4 β KO cells, but the immunoprecipitates from the KO cells rescued with wild-type β4 contained a similar amount of tepsin to immunoprecipitates from the wild-type control cells. A small amount of tepsin was detected in the immunoprecipitates from the KO cells rescued with mutant β4. B) Western blots of immunoprecipitates of AP4 β from extracts of HeLa cells stably expressing either wild-type or mutant tepsin-GFP (L470S/F471S), probed with antibodies against GFP, AP4 β and ε. Mutant tepsin-GFP co-immunoprecipitates with AP4 less than wild-type tepsin-GFP. C) Immunofluorescence double labeling for AP4 ε and tepsin in AP4 β KO HeLa cells stably rescued with wild-type or mutant (earless, Y682V or I669A/A670S) β4. Tepsin co-localizes with AP4 in the perinuclear region in all four rescued cell lines. Scale bars are 20 µm.
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