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First published online 14 February 2006
doi: 10.1242/jcs.02808

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Acidic clusters target transmembrane proteins to the contractile vacuole in Dictyostelium cells

¹ Université de Genève, Centre Médical Universitaire, Département de Physiologie Cellulaire et Métabolisme, CH-1211 Genève 4, Switzerland
² IFR 128 BioSciences Lyon-Gerland, Institut de Biologie et Chimie des Protéines, UMR5086–CNRS/Université Lyon I, 7 Passage du Vercors, 69367 Lyon CEDEX 07, France

View larger version (40K): [in a new window]   Fig. 1. The last 91 residues of Rh50 are sufficient for membrane protein localization on the CV. (A) Schematic representation of the CsA-Rh50 chimeric protein used in this study. The fusion protein is composed of the contact site A external domain (EX), P29F8 transmembrane domain (TM) and a short cytoplasmic domain (CYT) either followed by a stop codon (CsA-Stop) or the 91 C-terminal residues of Rh50 (pFL674). (B) Confocal microscopy study of cells expressing CsA-Stop or CsA-Rh50 (pFL674) constructs. Cells were double-labeled with the anti-CsA antibody (41-71-21) and polyclonal antibodies to either calmodulin or Rh50 (RH153) or the monoclonal antibody to p80 (H161) directly coupled to Alexa Fluor 488. Merged images are shown in the right-hand column. For calmodulin staining, cells were not fixed in paraformaldehyde but directly incubated in –20°C methanol for 10 minutes. CsA-Rh50 colocalized with endogenous Rh50 or with calmodulin, two CV-resident proteins, but was excluded from early and late p80-positive endosomes. Bar, 10 µm.

View larger version (26K): [in a new window]   Fig. 2. Ph50 has three putative sorting signals already described in other proteins. (A) Alignment of the Rh50 protein sequence with diaromatic signals characterized in the cation-dependant mannose 6P receptor (CD-MPR), the human mannose receptor (MR) and the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-env). Aromatic residues are underlined. (B) Alignment of the two acidic clusters found in Rh50 (Rh50a and Rh50b) with known acidic signals found in furin, HIV-1 Nef and yeast Ent3p. Acidic residues are underlined and the conserved F residue adjacent to acidic clusters is in bold.

View larger version (17K): [in a new window]   Fig. 3. Mutations introduced into the cytoplasmic domain of the CsA-Rh50 fusion protein. Positions of the mutations are indicated on the black line representing the cytoplasmic domain of the constructs. Each construct was given a name as indicated to the left of the figure. For pFL905, the sequence in bold corresponds to the last C-terminal acidic signal of Rh50.

View larger version (43K): [in a new window]   Fig. 4. Rh50 acidic clusters are sufficient for CV targeting. Dictyostelium cells expressing the indicated construct were double-labeled with the anti-CsA antibody (41-71-21) and the polyclonal antibody to calmodulin and analyzed by confocal microscopy. Although a domain containing only the diaromatic signal (pFL760) did not allow the transport of the CsA-Rh50 mutant to the CV, domains containing one (constructs pFL759 and pFL786) or two acidic signals (pFL761) were sufficient for targeting to the CV. Mutation of the acidic residues to alanine (pFL778) prevents the expression of CsA-Rh50 to the CV. Bar, 10 µm.

View larger version (58K): [in a new window]   Fig. 5. Transport of CsA-Rh50 from the plasma membrane to the CV. Cells expressing pFL759 were incubated with the anti-CsA antibody for 5 minutes at 4°C, washed and then warmed up for the times indicated on the left. The amount of CsA-Rh50 left on the cell surface after internalization was monitored by incubating cells at 4°C for 30 minutes with an anti-mouse secondary antibody coupled to Alexa Fluor 647 (CsA Surface, blue staining). Then, cells were fixed, methanol-permeabilized and incubated with an anti-mouse secondary antibody coupled to Alexa Fluor 568 to detect internalized CsA-Rh50 as well as any remaining construct on the cell surface (CsA Total, red staining). The CV compartment was decorated with a polyclonal antibody to Rh50 (RH153, green staining). Cells were observed by confocal microscopy. CsA-Rh50 was rapidly internalized, accumulated in vesicular structures and after 60 minutes of internalization localized on the CV network. Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 6. Internalized CsA-Rh50 is excluded from p80-containing endosomes and from the Golgi apparatus. (A) Cells expressing pFL759 were treated as described in Fig. 5. At the end of the procedure, early and late endosomal vacuoles were labeled with the monoclonal antibody to p80 (H161) directly coupled to Alexa Fluor 488 (p80, green staining). CsA-Rh50 barely colocalized with p80 endosomes even after 2 hours of internalization. Note that the CV compartment was not detected here upon staining with the anti-CsA antibody because p80 endosomes and the CV localize in different areas of the cell and that we selected by confocal microscopy a plane appropriate for the observation of p80 endosomes but excluding the CV network. (B) The Golgi marker golvesin (C)-GFP was expressed in cells expressing pFL759. Cells were treated as described in Fig. 5. After 5 minutes of internalization, CsA-Rh50 did not colocalize in golvesin (C)-GFP positive structures. Bar, 10 µm.

View larger version (60K): [in a new window]   Fig. 7. The acidic cluster is required for transport of CsA-Rh50 from the plasma membrane to the CV. Cells expressing pFL775 (A) or pFL778 (B) were treated as described in Fig. 5. Mutation of the FW signal to alanine residues (pFL775) did not affect the transport of the chimera to the CV whereas mutation of the acidic cluster to alanine (pFL778) inhibited CV targeting. Bar, 10 µm.

View larger version (18K): [in a new window]   Fig. 8. Rh50 acidic clusters interact with the ear domain of AP-1. The ability of the Rh50 cytoplasmic domain to interact with the -ear domain of AP-1 was tested in a yeast two-hybrid assay. Interaction of proteins was determined by measuring ß-galactosidase activity in liquid conditions. Results, expressed in arbitrary units, correspond to the average of three independent experiments. The s.e.m. was less than 5%. Both Ent3p and Rh50 proteins comparably interacted with the -ear domain of AP-1 in this assay but not with the µ1 subunit of the AP complex. Notably, mutation of either acidic cluster to alanine residues led to a dramatic reduction of the interaction between the -ear domain and Rh50.

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