Volume 7, Issue 4, Pages 619-625 (October 2004) An Ear-Core Interaction Regulates the Recruitment of the AP-3 Complex to Membranes  Stephane Lefrançois, Katy Janvier, Markus Boehm, Chean Eng Ooi, Juan S. Bonifacino  Developmental Cell  Volume 7, Issue 4, Pages 619-625 (October 2004) DOI: 10.1016/j.devcel.2004.08.009

Figure 1 δ-Ear Interacts with σ3A and σ3B (A) Schematic representation of AP-3. (B) Yeast cells cotransformed with the ear domains indicated on the left and the σ subunits indicated on top were tested for interactions by growth on plates with (+His) or without (−His) histidine. p53 and the SV40 large T antigen (LT) were used as controls. (C) GST, or GST-tagged δ-, γ1-, γ2-, and αC-ears bound to glutathione-Sepharose beads were incubated with HeLa cell lysate. Bound AP-3 was detected by immunoblotting with antibodies to β3 and σ3 (σ3 migrates as doublet, as previously shown by Dell'Angelica et al., 1997a). The first lane shows 10% of the input of HeLa cell lysate. The amount of protein loaded on each lane was verified using Ponceau staining (not shown). Developmental Cell 2004 7, 619-625DOI: (10.1016/j.devcel.2004.08.009)

Figure 2 The Carboxy-Terminal Extension of σ3A and σ3B Is Required for Interaction with δ-Ear (A) Schematic representation of the constructs used in yeast two-hybrid experiments. (B) Yeast cells cotransformed with full-length σ3A or σ3B, or σ3A (1–165) or σ3B (1–165) were tested for interaction with δ-ear (946–1153), δ-hinge (583–945), and δ-trunk (1–582) on plates with (+His) or without (−His) histidine. (C) Fragments of σ3A or σ3B (165–193 and 150–193) tagged with the HA epitope were tested for interaction with δ-ear as above. (D) Cell lysates prepared from yeast cells cotransformed as in (C) were analyzed by immunoblotting with anti-HA antibody. (E) Yeast cotransformed with σ1/σ3 chimeras (full-length σ1 appended with residues 165–193 of σ3A or σ3B) were tested for interaction with δ-ear. (F) The σ1/σ3 chimeras were tested for interaction with full-length γ1. Developmental Cell 2004 7, 619-625DOI: (10.1016/j.devcel.2004.08.009)

Figure 3 δ-Ear Blocks the Interaction of AP-3 with Arf1 but Not with Dileucine Signals (A) GST-tagged LIMP-II bound to GST beads was incubated with HeLa cell lysate in the presence of 0, 1, 5, or 10 μg of His6-tagged δ-ear. The first lane corresponds to 10% of the input of HeLa cell lysate. (B) His6-tagged Arf1Q71L, wild-type Arf1, and Arf1T31N were bound to Ni-NTA and incubated with HeLa cell lysate. Bound AP-3, AP-1, and AP-2 and His6-tagged Arf proteins were detected by immunoblotting with anti-β3, anti-γ1, anti-μ2, and anti-His6, respectively. Shown is a representative immunoblot from three experiments. (C) Yeast cells were cotransformed with plasmids encoding δ or ϵ and pBridge encoding various Arf1 cDNAs in MCS I and σ3A, σ3B, or σ4 in MCS II. Transformants were grown on plates with (+His) or without (−His) histidine and without methionine (−Met). (D) Yeast cells were cotransformed with pGADT7 encoding δ-ear or δ-trunk and pBridge encoding Arf1Q71L in MCS I and either σ3A or σ3A1-165 in MCS II. Transformants were grown on plates with (+His) or without (−His) histidine and without methionine (−Met). (E) His6-tagged Arf1Q71L was bound to Ni-NTA and incubated with HeLa cell lysate in the presence of 0, 1, 5, or 10 μg of GST-δ-ear, GST-γ1-ear, or GST-ϵ-ear. Bound AP-3 and AP-1 and His6-tagged Arf proteins were detected by immunoblotting with anti-β3, anti-γ1, and anti-His6, respectively. Developmental Cell 2004 7, 619-625DOI: (10.1016/j.devcel.2004.08.009)

Figure 4 δ-Ear Interferes with AP-3 Recruitment to Membranes (A) In vitro experiments. Endosomal membranes were incubated with HeLa cell lysate that was preincubated with (+) or without (−) 10 μg His6-δ-ear (4.5 μM) in the presence of an ATP regenerating system with (+) or without (−) 3 μM GTPγS. Membrane bound AP-3, AP-1, and AP-2 were detected by immunoblotting with anti-β3, anti-γ1, and anti-μ2, respectively. (B) Membranes were treated as in (A), except that HeLa cell lysate was preincubated with (+) or without (−) 10 μg of His6-ϵ-ear or 10 μg of His6-γ1-ear. (C–K) δ-ear prevents recruitment of AP-3 to membranes in vivo. COS-7 cells overexpressing myc-tagged δ-ear (red; D, E, G, H) or myc-tagged ϵ-ear (red; J, K) were stained with anti-myc antibody and anti-δ (green; C, E, I, K) or anti-γ1 (green; F, H). Scale bar equals 10 μm. (L) Increased surface expression of the Lamps in cells depleted of μ3 using a specific siRNA. Results are the mean ± SD from three separate experiments. (M) Overexpression of δ-ear causes increased surface expression of Lamps in HeLa cells. Surface expression of Lamp-1 and CD63 in HeLa cells overexpressing the myc epitope (i.e., vector), myc-tagged δ-ear, and myc-tagged ϵ-ear was analyzed by flow cytofluorometry. Results are the mean ± SD from five separate experiments. *p < 0.005; **p < 0.05. (N) Schematic representation of the regulation of AP-3 recruitment to membranes by δ-ear-σ3 interactions. Developmental Cell 2004 7, 619-625DOI: (10.1016/j.devcel.2004.08.009)