Structure of stonins and related proteins. (A) Schematic representation of human stonin 1 (Hs Stn1, GenBank/EMBL/DDBJ accession no. AF255310), human stonin 2 (Hs Stn2, AF255309), D. melanogaster stoned B (Dm STNB, U54982), C. elegans hypothetical protein C27H6.1 (Ce C27H6.1, Z81042), and the human μ2 subunit of the AP-2 complex (Hs μ2, NM_004068). Specific domains of homology are color coded and denoted as follows: PRD, proline-rich domain; SHD, stonin homology domain; MHD, μ-homology domain; BBD, β-binding domain. Black lines denote regions with no significant sequence homology. Numbers of NPF or DPF motifs in the PRDs are indicated. (B) Alignment of short sequences in the PRD of human stonin 2 with homologous sequences in human (Hs) epsin 1 (AF073727), epsin 2 (AF062084), and amphiphysin 2 (AAB63263). Identical residues are boxed, and NPF motifs are highlighted in yellow. (C) Multiple sequence alignment of the SHD and MHD domains of stonin family members. The black and gray boxes indicate identical and strongly conserved amino acids, respectively. Blue and orange dots indicate amino acids in μ2 that are involved in binding YXXØ-type signals (Owen and Evans 1998) and the C2B domain of synaptotagmin I (Haucke et al. 2000), respectively. Numbers denoting the last amino acid in each sequence are indicated on the right.

Interactions of stonin 2 (Stn2) with Eps15 and intersectin 1 analyzed using the yeast two-hybrid system (A) and in vitro binding assays (B–D). (A) GAL4 DNA binding domain constructs encoding the three EH domains from Eps15 or the related Eps15R protein, the two EH domains from intersectin 1, or mouse (m) p53 (as a specificity control) were coexpressed with GAL4ad constructs encoding full-length stonin 1, full-length stonin 2, the PRD + SHD domains of stonin 2 (Fig. 1 A) or various mutants of this segment, epsin 2, or the SV40 T antigen (as a specificity control). Interactions were tested by monitoring growth on plates containing –His +Ade, –His –Ade, or +His +Ade (control) media. (B) In vitro translated, [³⁵S]methionine-labeled PRD + SHD domains of stonin 2 tagged with the Myc epitope and mutants of this construct were tested for interaction with either a GST fusion protein bearing the three EH domains of Eps15 or with GST using a conventional pull-down assay. Fractions bound and not bound to the glutathione–Sepharose beads are shown. (C) Similar to B, except that stonin 2 constructs were expressed by transient transfection into HeLa cells, extracted with Triton X-100, and bound and unbound proteins detected by immunoblotting with anti-Myc antibody. (D) A Triton X-100 extract of [³⁵S]methionine-labeled HeLa cells containing endogenous stonin 1 and stonin 2 was incubated with GST or GST fusion proteins bearing the three EH domains from Eps15 or the two EH domains from intersectin 1. Unbound and bound stonin 1 and stonin 2 were isolated by immunoprecipitation–recapture. The positions of molecular mass markers (in kD) are indicated on the left of B–D.

Interactions of stonin 2 with Eps15, AP-2, and intersectin 1 in vivo. (A) Coprecipitation of endogenous stonin 2 with Eps15. A Triton X-100 extract of HeLa cells was subjected to immunoprecipitation with preimmune sera (PI) or antibodies to stonin 1 (Stn1) or stonin 2 (Stn2). The presence of coprecipitated Eps15 was detected by immunoblotting. (B) Coprecipitation of transfected GFP–stonin 2 with endogenous intersectin 1. The GFP or GFP–stonin 2 constructs were expressed by transfection into HeLa cells and isolated by immunoprecipitation with anti-GFP or antiintersectin antibodies from Triton X-100 extracts of the cells. The presence of the GFP proteins was revealed by immunoblotting using anti-GFP antibodies. (C) Coprecipitation of endogenous Eps15 and AP-2 with transfected GFP–stonin constructs. The GFP–stonin fusion proteins indicated in the figure were expressed by transfection into HeLa cells and isolated by immunoprecipitation with anti-GFP antibodies from Triton X-100 extracts of the cells. The presence of Eps15 and the α subunit of AP-2 were revealed by immunoblotting. The positions of molecular mass markers (in kD) are indicated on the left. *Indicates the heavy chains of anti-GFP antibody used in B and C.

Effects of overexpression of GFP–stonin 2 on Cy3-Tf, rhodamine-EGF (Rhod-EGF), Dil-LDL internalization, and AP-2 localization. HeLa cells were transfected with plasmids encoding either GFP–stonin 2 (A–I and M–O) or GFP alone (J–L). Live cells were incubated for 15 min in the presence of Cy3-Tf (A–C and J–L), rhodamine-EGF (D–F) or Dil-LDL (G–I) at 37°C. AP-2 (M–O) was detected by immunofluorescence microscopy using antibodies to α-adaptin (AP.6) followed by Cy3-conjugated donkey anti–mouse IgG. Cells were examined by confocal fluorescence microscopy. (A, D, G, J, and M) GFP fluorescence (green); (B and K) Cy3-Tf (red); (E) Rhodamine-EGF (red); (H) Dil-LDL (red); (N) AP-2 (red). Arrow points to transfected cell exhibiting accumulation of AP-2 in the juxtanuclear area. Bars, 10 μm.

Expression of stonins in human tissues and cells. (A) Northern blot analysis of the expression of stonin 1 (Stn1) and stonin 2 (Stn2) mRNAs in human tissues. The positions of RNA molecular size markers (in kb) are indicated on the left. (B) Immunoprecipitation–recapture of endogenous stonin 1 and stonin 2 proteins from [³⁵S]methionine-labeled HeLa cells using preimmune serum (PI), specific antibodies to each protein, or specific antibodies preabsorbed with the immunogens. Immunoprecipitates were analyzed by 4–20% SDS-PAGE. The positions of protein molecular mass markers (in kD) are indicated on the left. (C) Expression of endogenous stonin 1 and stonin 2 proteins in different human cell lines. Stonins were isolated from equal quantities of [³⁵S]methionine-labeled cell lines using specific antibodies to each protein, as in B.

Analysis of the association of stonins with membranes. (A) Immunoprecipitation–recapture of stonin 1 (Stn1) and stonin 2 (Stn2) from a postnuclear supernatant (PNS) of [³⁵S]methionine-labeled HeLa cells and from cytosolic (C) and membrane (M) fractions of the PNS after ultracentrifugation. (B) Membrane fractions obtained as in A were extracted for 45 min at 4°C with the solutions indicated on top. Pellet (P) and supernatant (S) fractions were then prepared by ultracentrifugation, and stonin 1, stonin 2, and lamp-1 were isolated by immunoprecipitation–recapture with specific antibodies. (C) [³⁵S]methionine-labeled HeLa cells were solubilized in 2% (wt/vol) Triton X-100 on ice for 30 min, and the extract was centrifuged on a discontinuous sucrose gradient. Stonin 2, caveolin, the γ1 subunit of AP-1 and lamp-1 were then isolated from the indicated gradient fractions using specific antibodies. The positions of molecular mass markers (in kD) are indicated on the left. (D) Analysis of the size of stonins by sedimentation velocity. A cytosolic fraction of [³⁵S]methionine-labeled HeLa cells was separated by ultracentrifugation on a 4–20% sucrose gradient. Stonin 1, stonin 2, and γ1-adaptin (a marker for the position of cytosolic AP-1, 7.7S; Nakagawa et al. 2000) were isolated by immunoprecipitation–recapture from gradient fractions.

Stonin 2 binds to synaptotagmins I and II. (A) Interaction of stonin 2 with the cytoplasmic (C2AB) domains of synaptotagmins I and II. Triton X-100 extracts of HeLa cells, transiently expressing Myc-Stn2 or from untransfected cells, were incubated with GST fusion proteins comprising the C2A and C2B domains of synaptotagmins I or II, or with GST–GGA3_GAE (specificity control), using a conventional pull-down assay. (B) Interaction of stonin 2 with the C2B domain of synaptotagmins I and II. Similar to A, except that the cell extracts were incubated with GST fusion proteins comprising the C2A or C2B domains of synaptotagmins I and II. The presence of Myc-Stn2 in the bound material was revealed by immunoblotting (IB) using monoclonal antibodies to the Myc epitope. Coomassie blue staining of the gels revealed that similar amounts of GST fusion proteins were used.