J Biol Chem. 2012 May 18;287(21):17398-407. doi: 10.1074/jbc.M112.349035. Epub 2012 Mar 28.

Adaptor autoregulation promotes coordinated binding within clathrin coats.

Hung CW, Aoh QL, Joglekar AP, Payne GS, Duncan MC.

Source

Department of Biology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina 27599, USA.

Abstract

Membrane traffic is an essential process that allows protein and lipid exchange between the endocytic, lysosomal, and secretory compartments. Clathrin-mediated traffic between the trans-Golgi network and endosomes mediates responses to the environment through the sorting of biosynthetic and endocytic protein cargo. Traffic through this pathway is initiated by the controlled assembly of a clathrin-adaptor protein coat on the cytosolic surface of the originating organelle. In this process, clathrin is recruited by different adaptor proteins that act as a bridge between clathrin and the transmembrane cargo proteins to be transported. Interactions between adaptors and clathrin and between different types of adaptors lead to the formation of a densely packed protein network within the coat. A key unresolved issue is how the highly complex adaptor-clathrin interaction and adaptor-adaptor interaction landscape lead to the correct spatiotemporal assembly of the clathrin coat. Here we report the discovery of a new autoregulatory motif within the clathrin adaptor Gga2 that drives synergistic binding of Gga2 to clathrin and the adaptor Ent5. This autoregulation influences the temporal and/or spatial location of the Gga2-Ent5 interaction. We propose that this synergistic binding provides built-in regulation to ensure the correct assembly of clathrin coats.

PMID:: 22457357; [PubMed - indexed for MEDLINE]
PMCID:: PMC3366796

Free PMC Article

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FIGURE 1.

Gga2 interaction with clathrin is altered in adaptor deletion strains. Clathrin was immunoprecipitated from the indicated strains with a polyclonal clathrin light chain antibody, and interacting Ent5 and Gga2 were detected by immunoblot analysis. The top panel shows a representative immunoprecipitation (I.P.) reaction. The bottom panel shows data from four independent immunoprecipitation experiments. Intensity measurements of adaptors were normalized to intensity in wild-type (wt) sample of the same experiment and then normalized to the intensity of clathrin heavy chain bands (Chc1) within the same sample. *, p value <0.05 as determined by a two-tailed Student's t test.

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

J Biol Chem. 2012 May 18;287(21):17398-17407.

FIGURE 2.

Mutations of the clathrin box region alter interaction of Gga2 with clathrin in vitro. a, top panel, schematic of Gga2 and interacting partners. Black dotted lines indicate previously defined interactions. The green dotted line indicates an intramolecular interaction defined in this study. Middle panel, schematic of fragments used in in vitro assays. Bottom panel, schematic of mutation generated. b, indicated mutations were generated in GST fusion constructs containing 60 amino acids of the Gga2 hinge, purified, and incubated with purified clathrin. Interacting clathrin was detected by immunoblot analysis. c, indicated fusion proteins were incubated with cell lysates, and interacting clathrin was detected by immunoblot analysis. d, indicated mutations were generated in GST fusion constructs containing the Gga2 hinge and ear, purified, and incubated with cell lysates. Interacting clathrin was detected by immunoblot analysis. wt, wild type.

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

J Biol Chem. 2012 May 18;287(21):17398-17407.

FIGURE 3.

Mutations of the clathrin box region alter interaction of Gga2 with clathrin in cell lysates. a, effect of Gga2 mutations on clathrin binding to Gga2 and Ent5 in lysates. The indicated mutations were generated at the endogenous Gga2 locus. Clathrin was immunoprecipitated from indicated strains with a polyclonal clathrin light chain antibody, and interacting Gga2 and Ent5 were detected by immunoblot analysis. The left panel shows representative immunoprecipitation (I.P.) results. The right panels show intensity analysis as described in Fig. 1. b, effect of Gga2 mutations on Gga2 binding to clathrin and Ent5 in lysates Indicated mutations were generated at the endogenous Gga2 locus and GFP-tagged at the C terminus. Gga2-GFP alleles were immunoprecipitated with a monoclonal GFP antibody, and interacting clathrin and Ent5 were detected by immunoblot analysis.

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

J Biol Chem. 2012 May 18;287(21):17398-17407.

FIGURE 4.

The clathrin box region of Gga2 interacts with the Gga2 γ-ear. a, the clathrin box region binds to the γ-ear. A His₆-tagged 60-amino acid fragment containing the wild-type (CAB-wt) or a Δcore (CAB-Δcore) clathrin box region incubated with a GST-tagged fragment encoding the γ-ear (γ-ear) were incubated with or without cross-linker. Identical samples were processed for Western blot analysis and Coomassie staining. Note that un-cross-linked CAB-wt does not adhere to membrane in the immunoblot but is present in the Coomassie-stained gel. The arrowhead indicates migration of a band present only in reactions containing the CAB, γ-ear, and cross-linker. b, a fragment containing the Δcore mutant adopts an extended conformation. Elution profiles of Gga2 amino acid 340 to the end and Gga2 Δcore mutant amino acid 340 to the end in gel filtration are shown. Weight-averaged solute molar mass for the wild-type fragment was 25.18 kDa, and for the mutant fragment was 26.02 kDa with an error of 1% for both mass values.

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

J Biol Chem. 2012 May 18;287(21):17398-17407.

FIGURE 5.

CAB mutations alter colocalization of Ent5 and Gga2. a, representative images of cells analyzed. The indicated mutations were generated at the endogenous Gga2 locus and GFP-tagged at the C terminus, and Ent5 tagged at the C terminus with mCherry was introduced into these strains through crosses. The cells were imaged as described under “Experimental Procedures.” b, colocalization analysis of Gga2 alleles with Ent5. The box plot diagrams show the Manders' coefficients for analyzed data. On each box, the central mark is the median, the edges of the box are the 25^th and 75^th percentiles, the whiskers extend to the 95^th percentile, and the outliers are plotted individually. The outliers are indicated as circles. p values reflect a Mann-Whitney test with Null hypothesis (h = 0) that the two samples have the identical median with 95% confidence interval. *, p value greater than 1 × 10⁻³ for samples compared with wild type.

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

J Biol Chem. 2012 May 18;287(21):17398-17407.

FIGURE 6.

Clathrin box region mutations alters the recruitment differential between Gga2 and Ent5. a, representative images of events analyzed. The green arrow indicates the time point utilized for t = 0. The red arrow indicates the time point used to calculate the differential between Gga2 and Ent5 recruitment. The black arrow indicates the time point used to calculate event life span. Frames of time lapse are 2 s apart for each channel. Approximately 800 ms separates channels at a single time point. b, box plot diagrams of analyzed recruitment differential data. c, box plot diagrams of analyzed life span data. On each box, the central mark is the median, the edges of the box are the 25^th and 75^th percentiles, the whiskers extend to the 95^th percentile, and the outliers are plotted individually. The outliers are indicated as circles. p values reflect a Mann-Whitney test with Null hypothesis (h = 0) that the two samples have the identical median with 95% confidence interval. wt, wild type.

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

J Biol Chem. 2012 May 18;287(21):17398-17407.

FIGURE 7.

Schematic of possible adaptor recruitment models. a, different types of adaptor vesicle form in a temporal order on an organelle. Event type 1, which utilizes only Gga2 (orange) forms early on an organelle. Later in the life span of the same organelle, event type 2 vesicles form, which utilizes both Gga2 and Ent5 (purple). Finally in late stage organelles, only event type 3 vesicles form, which contain Ent5 and AP-1 (blue). Mutations of the autoregulatory domain of Gga2 may cause the loss of event type 1, resulting in the observed increase in Ent5 and Gga2 colocalization and the loss of temporal order of recruitment between Gga2 and Ent5. b, model of possible role of CAB in Ent5 recruitment in event type 2 vesicles. Gga2 in its autoregulated form becomes concentrated at a cellular membrane. DLL-type motifs of Gga2 loosely recruit clathrin. Now in local high concentration, clathrin can bind to the CAB, thus promoting the open form of Gga2. In the open form, Gga2 helps to stabilize Ent5 in the clathrin coat.

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

J Biol Chem. 2012 May 18;287(21):17398-17407.

Publication Types, MeSH Terms, Substances, Grant Support

Publication Types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH Terms

Adaptor Proteins, Vesicular Transport/genetics
Adaptor Proteins, Vesicular Transport/metabolism*
Amino Acid Motifs
Biological Transport, Active/physiology
Clathrin/genetics
Clathrin/metabolism*
Endosomes/genetics
Endosomes/metabolism
Saccharomyces cerevisiae/genetics
Saccharomyces cerevisiae/metabolism*
Saccharomyces cerevisiae Proteins/genetics
Saccharomyces cerevisiae Proteins/metabolism*
trans-Golgi Network/genetics
trans-Golgi Network/metabolism*

Substances

Adaptor Proteins, Vesicular Transport
Clathrin
Ent5 protein, S cerevisiae
Gga2 protein, S cerevisiae
Saccharomyces cerevisiae Proteins

Grant Support

GM092741/GM/NIGMS NIH HHS/United States
GM39040/GM/NIGMS NIH HHS/United States
R01 GM092741/GM/NIGMS NIH HHS/United States

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Adaptor autoregulation promotes coordinated binding within clathrin coats.

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