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First published online 15 January 2008
doi: 10.1242/jcs.012708

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ENTH domain proteins are cargo adaptors for multiple SNARE proteins at the TGN endosome

Subbulakshmi Chidambaram^*, Jana Zimmermann and Gabriele Fischer von Mollard

Biochemie III, Fakultät für Chemie, Universitätstrasse 25, Universität Bielefeld, 33615 Bielefeld, Germany

View larger version (62K): [in this window] [in a new window]   Fig. 1. The N termini of endosomal SNAREs interact with the ENTH domain of epsinR and Ent3p. (A) Two-hybrid interactions were detected by the ability of yeast cells (L40) to grow on selective plates. A fusion of the DNA-binding domain of LexA and the N terminus of Vti1b (amino acids 1-128, pBK111), syntaxin 7 (amino acids 1-161, pBK193) and syntaxin 8 (amino acids 1-142, pBK179) interacted with ENTH domain (amino acids 1-162) of epsinR fused to the VP16 activation domain (pBK130). No interaction was found of epsinR with the VP16 alone (pVP16-3). (B) Two-hybrid interactions were detected between the ENTH domain of Ent3p (amino acids 1-172, VP16 fusion, pKW3) and Vti1p (amino acids 1-115, LexA fusion, pBK118), Pep12p (amino acids 1-200, pBK171) and Syn8p (amino acids 1-169, pBK165) but not with Ykt6p (amino acids 1-140, pNK4), Tlg1p (amino acids 1-137, pBK172) or Vam7p (amino acids 1-255, pJZ6). ENTH domain of Ent5p (amino acids 1-172, pBK160) did not interact with any of the SNAREs. (C) In vitro binding of a bacterially expressed protein consisting of the ENTH domain of Ent3p (amino acids 1-172) fused to a C-terminal Strep-tag (pKW5) to His6-Vti1p (amino acids 1-194, pFvM112) and His6-Pep12p (amino acids 1-268, pFvM135), but not to His6-Tlg1p (amino acids 1-137, pFN3) or His6-Syn8p (amino acids 1-169, pFN6). (D) Weak in vitro binding of the ENTH domain of epsinR (amino acids 1-162) fused to a C-terminal Strep-tag (pNM3) to His6-syntaxin 7 (amino acids 1-161, pTW1) and His6-syntaxin 8 (amino acids 1-142, pTW2). Negative control: His6-Vti1a (amino acids 1-187, pBK39), positive control: His6-Vti1b (amino acids 1-207, pBK38).

View larger version (42K): [in this window] [in a new window]   Fig. 2. Genetic interactions between SYN8, ENT3 and ENT5. (A) The absence of Ent3p and Ent5p led to a synthetic growth defect in syn8 cells at 30°C and 37°C. (B) Absence of Ent3p and Ent5p resulted in synthetic defects of CPY transport to the vacuole in syn8 cells. CPY was immunoprecipitated from cellular extracts (I) and the medium (E) after pulse-chase labeling at 30°C. p1CPY, endoplasmic reticulum pro1CPY; p2CPY, Golgi pro2CPY; mCPY, vacuolar mature CPY. (C) Quantification of CPY secretion. Data for WT, ent3 and ent5 cells were from a previous experiment. n=2, Bars indicate range of values. Strains used in this figure were BY4742 (WT) and derivates: syn8, JZY1, JZY3, JZY2, ent3, ent5, BKY13

View larger version (27K): [in this window] [in a new window]   Fig. 3. Pep12p was more stable in the absence of Ent3p. Cells were labeled with [35S]methionine for 25 minutes and chased for 10 minutes, 3 hours or 5 hours. Pep12p was immunoprecipitated from the cellular extracts and the percentage of Pep12p remaining after 3 and 5 hours was calculated. Strains used: SEY6210, SCY2, SCY25, SCY26

View larger version (45K): [in this window] [in a new window]   Fig. 4. Ent3p and Ent5p were not involved in transport of all cargo proteins between the TGN and endosomes. Retrograde transport from the late endosome to the TGN was monitored using A-ALP (A) and the CPY receptor Vps10p (B). Both proteins contain a retrieval signal for recycling from the late endosome to the TGN. A-ALP consists of the cytosolic domain of dipeptidyl aminopeptidase A (DPAP A), which localizes to the TGN, fused to the transmembrane and luminal domain of alkaline phosphatase (ALP) for detection (Nothwehr et al., 1993). The stability of these proteins was not strongly reduced indicating that their trafficking was not significantly affected by the lack of Ent3p or Ent5p [The percentage of A-ALP remaining compared to the amount after the 5 minutes chase: 60 minutes chase, WT 68% (s.d. ±24, n=3), ent3 65% (s.d. ±14), ent5 56% (s.d. ±10) and 120 minutes chase, WT 39% (s.d. ±17), ent3 35% (s.d. ±15), ent5 24% (s.d. ±17). The percentage of intact Vps10p compared to total Vps10p after 3 hours of chase was: WT 58% (±2.2%, n=2), ent3 69% (±0.9%), ent5 65% (±0.5%) and ent3 ent5 61% (±0.3%).] Cells were pulse labeled at 37°C and chased for the indicated time periods before immunoprecipitation. Vps10p was immunoprecipitated with antisera directed against Vps10p from strains SEY6211, SCY2, SCY25 and SCY26. A-ALP was immunoprecipitated with antisera directed against ALP in cells deleted for PHO8, the gene encoding ALP to avoid immunoprecipitation of ALP (strains: SNY18, BKY25, BKY26 all with the CEN plasmid pSN55 encoding A-ALP). pA-ALP, Golgi proA-ALP; mA-ALP, mature A-ALP processed in the vacuole; Vps10p*, Vps10p cleaved in the vacuole.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Pep12p did not colocalize with Ent3p in wild-type cells and was more diffuse in ent3 ent5 and syn8 ent3 ent5 cells. Wild-type (SEY6210, A-C; BY4742, D,G), ent3 ent5 (BKY13, E, H) and syn8 ent3 ent5 (JZY2, F,I) cells were grown at 30°C and immunofluorescence was done using antisera against Pep12p (A,C-F) and Ent3p (B,C). Cells grown at 30°C were incubated with FM4-64 for 5 minutes to stain endosomal structures (G-I). The staining pattern was unchanged, indicating that endosomes were intact in mutant cells. Bar, 5 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Pep12p and Vti1p were redistributed to denser fractions in ent3 ent5 and syn8 ent3 ent5 cells on a sucrose density gradient. Cleared yeast homogenates from wild-type (BY4742), syn8, ent3 ent5 (BKY13) and syn8 ent3 ent5 (JZY2) cells were fractionated on a 19% to 42% sucrose density gradient and analyzed by immunoblotting for Pep12p (A) and Vti1p (B). The vacuolar 60 kDa ATPase (C) was present in the vacuolar fractions (3-5) and ER was found in fractions 9-11 as indicated by the marker Use1p (D).

View larger version (37K): [in this window] [in a new window]   Fig. 7. Interaction between Ent3p and Pep12p was dependent on an intact FSD motif in Pep12p. (A) Yeast two-hybrid interactions were determined in L40 cells expressing the ENTH domain of Ent3p as VP16 activation domain fusion (pKW3) together with fusions of LexA DNA binding domain with the N terminus of wild-type Pep12p (AA 1-200, pBK171), with Pep12p (AA 1-200) carrying a deletion of amino acid residues 19-26 (FSD, pJZ10), or with Pep12p (AA 1-200) with the amino acid substitution F20L (pJZ9), respectively. Growth on selective plates containing 5 mM 3-aminotriazole was analyzed. Expression of VP16 activation domain served as negative control. (B) In vitro pull-down assay of a bacterially expressed protein consisting of the ENTH domain of Ent3p (amino acids 1-172) fused to a C-terminal Strep-tag (pKW5). His6-Pep12p (amino acids 1-268) with the amino acid substitution F20L (pSK3) or carrying a deletion of amino acid residues 19-26 (FSD, pSK4) bound less Ent3-Strep than wild-type His6-Pep12p (amino acids 1-268, pFvM135). Some background binding to His6-Tlg1p (Tlg1p, amino acids 1-137, pFN3) was observed. Immunoblots were stained with antisera against Pep12p (top panel) or Ent3p (bottom panel) and quantified. The ratio of bound Ent3p to wild-type Pep12p was set to one.

View larger version (22K): [in this window] [in a new window]   Fig. 8. A defective FSD motif in Pep12p had similar effects on Pep12p stability as absence of Ent3p. Cells were labeled with [35S]methionine for 25 minutes and chased for 10 minutes, 3 hours or 5 hours. Pep12p was immunoprecipitated from the cellular extracts and the percentage of Pep12p remaining after 3 and 5 hours was calculated. Strains used: WT, SEY6210; ent3, SCY2; F20L, SCY27 (pep12::URA3) pJZ11 (CEN PEP12-F20L).

View larger version (43K): [in this window] [in a new window]   Fig. 9. Model for the role of Ent3p, epsinR and SNAREs in endosomal traffic. In yeast (upper panel), Ent3p is required for traffic from the TGN to late endosomes. The ENTH domain of Ent3p interacts with Vti1p, Pep12p and Syn8p, which form a SNARE complex together with Ykt6p in traffic from the TGN to the late endosome. Ent3p functions in cargo sorting of Pep12p in traffic from the TGN to late endosomes (dashed arrow). In mammalian cells, epsinR is involved in retrograde traffic from the early endosome to the TGN. EpsinR interacted with Vti1b, syntaxin 7 (Syx7) and syntaxin 8 (Syx8), which have been implicated in homotypic fusion of late endosomes in a complex with VAMP8 and with VAMP7 transport from late endosomes to lysosomes. It has not been studied whether epsinR also functions in TGN to late endosome traffic.