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1.
Fig. 4.

Fig. 4. From: Bat3 promotes the membrane integration of tail-anchored proteins.

A Bat3-containing fraction restores Sec61β-OPG membrane integration. (A) Reticulocyte lysate was incubated with different resins and the depletion of various cytosolic factors determined by immunoblotting, as indicated. (B) The resin-depleted lysate preparations were used to stimulate the membrane integration of recombinant Sec61βOPG. (C) Material bound to Cibacron Blue agarose was recovered and analysed for Bat3 content by immunoblotting and comparison with equivalent amounts of untreated lysate and Cibacron-depleted lysate as indicated. Lane 3 is empty. (D) The membrane integration of recombinant Sec61β-OPG was determined in the presence of untreated lysate (lane 1), Cibacron-depleted lysate supplemented with buffer (lane 2), or Cibacron-depleted lysate supplemented with the eluate from the Cibacron resin (lane 3). Individual boxes all represent a single exposure, as described for Fig. 3.

Pawel Leznicki, et al. J Cell Sci. 2010 July 1;123(13):2170-2178.
2.
Fig. 2.

Fig. 2. From: Bat3 promotes the membrane integration of tail-anchored proteins.

Identification of tail-anchor-specific cytosolic factors. (A) Comparable amounts of opsin epitope-tagged versions (OPG) of a Sec61β variant lacking the transmembrane domain (−TMD), full-length Sec61β and RAMP4 (supplementary material Fig. S1A) were immobilised on UltraLink Biosupport, incubated with rabbit reticulocyte lysate and binding partners eluted with 0.1% (v/v) Triton X-100 after extensive washing. Following SDS-PAGE and Coomassie Blue staining, components strongly enriched in lanes 2 and 3 were further characterised. Relevant proteins successfully identified by mass spectrometry are shown (*, **), other candidates (?) remain uncharacterised. (B) Eluted material was analysed by immunoblotting for specific components, as indicated.

Pawel Leznicki, et al. J Cell Sci. 2010 July 1;123(13):2170-2178.
3.
Fig. 7.

Fig. 7. From: Bat3 promotes the membrane integration of tail-anchored proteins.

Bat3 relocalisation of GFP-Sed5 is independent of Sgt2. (A) Reticulocyte-lysate components eluted from recombinant TA proteins with or without a TA segment (see Fig. 2) were analysed for the presence of SGTA by immunoblotting. (B) Live-cell imaging of GFP-Sed5 in wild-type, Δsgt2 and Δmdy2sgt2 S. cerevisiae expressing full-length human Bat3. (C) Summary of potential interactions between TA proteins, Bat3, SGTA/Sgt2, Hsp70 and components of the TRC40/GET pathway (see Chang et al., 2010; Corduan et al., 2009; Costanzo et al., 2010; Favaloro et al., 2008; Jonikas et al., 2009; Sasaki et al., 2008; Schuldiner et al., 2008; Stefanovic and Hegde, 2007; Stelzl et al., 2005; Winnefeld et al., 2006). Solid lines indicate known or presumed physical interactions, the dashed line, a regulatory interaction and the dotted lines indicate possible contributions of Bat3 to TA-protein biogenesis.

Pawel Leznicki, et al. J Cell Sci. 2010 July 1;123(13):2170-2178.
4.
Fig. 6.

Fig. 6. From: Bat3 promotes the membrane integration of tail-anchored proteins.

Bat3 relocalises GFP-Sed5 in S. cerevisiae GET mutants. (A) Outline of full-length Bat3 (isoform 2), and the N-terminal fragment used in this study. The locations of the ubiquitin-like domain, NLS and BAG domains and the antibody-binding region are indicated. In the ΔNLS mutant, the KRRK motif shown is altered to KRSL to disrupt nuclear targeting of Bat3 (Manchen and Hubberstey, 2001). (B) Immunoblot showing Bat3 and phosphoglycerate kinase 1 (Pgk1) levels in wild-type or Δmdy2 (Δget5)-transformed S. cerevisiae cells. (C) Subcellular localisation of full-length Bat3 expressed in wild-type S. cerevisiae and DAPI staining of nuclei visualised by immunofluorescence microscopy. (D-H) The effect of Bat3 expression upon the subcellular localisation of GFP-Sed5 was determined by live-cell imaging in wild-type, Δget3, Δget4 or Δmdy2get5) cells, as indicated. Full-length Bat3, an N-terminal fragment or the ΔNLS mutant, were used as indicated. See also supplementary material Fig. S2C for fixed and immunostained cells of the same genotype demonstrating co-localisation of Bat3 and GFP-Sed5 immunoreactivity with DAPI staining of the nucleus in Δmdy2get5) cells. Scale bar: 5 μm.

Pawel Leznicki, et al. J Cell Sci. 2010 July 1;123(13):2170-2178.
5.
Fig. 1.

Fig. 1. From: Bat3 promotes the membrane integration of tail-anchored proteins.

Sec61β-OPG membrane integration requires cytosol and nucleotide triphosphates. (A) A membrane-integration reaction of recombinant Sec61βOPG was performed in the presence of sheep microsomes and either rabbit reticulocyte lysate (RRL) or a buffer control. The membrane fraction was isolated and analysed by immunoblotting with a monoclonal antibody recognising the OPG tag. N-glycosylated (+gly) and non-glycosylated (−gly) forms of Sec61βOPG are labelled. The lower product is a truncated version of Sec61βOPG (supplementary material Fig. S1B,C). (B) Three 50 μl membrane-integration reactions containing ~1.1 μM Sec61β-OPG and ~2.1 OD280/ml sheep pancreatic microsomes supplemented with increasing amounts of rabbit reticulocyte lysate (% of total reaction volume shown) were performed and analysed by EndoH treatment and immunoblotting. (C) A standard membrane-integration reaction carried out with untreated rabbit reticulocyte lysate in the presence or absence of an energy regenerating system, as indicated.

Pawel Leznicki, et al. J Cell Sci. 2010 July 1;123(13):2170-2178.
6.
Fig. 5.

Fig. 5. From: Bat3 promotes the membrane integration of tail-anchored proteins.

Bat3 and TRC40 are associated with distinct populations of Sec61β-OPG polypeptides. (A) To recapitulate biosynthetic associations, Sec61β-OPG was translated in rabbit reticulocyte lysate, the material subjected to centrifugation through a 5-25% sucrose gradient, and 13 fractions plus the pellet recovered (fraction 1=top, p=pellet). Following SDS-PAGE, the location of Sec61βOPG chains was determined by phosphorimaging of the radiolabelled chains (upper panel), whereas the migration of various cytosolic factors was established by immunoblotting (as labelled). The distortion of the radiolabelled Sec61βOPG samples seen in fractions 4-8 results from the presence of large quantities of unlabelled globin chains present in the lysate used for in vitro translation. (B) A portion of each sucrose gradient fraction was incubated with canine pancreatic microsomes, the membranes recovered and the glycosylation status of Sec61β-OPG determined by phosphorimaging. The proportion of the total N-glycosylated material resulting from each individual fraction (% total glyc.) was estimated to provide a comparative measure of membrane integration.

Pawel Leznicki, et al. J Cell Sci. 2010 July 1;123(13):2170-2178.
7.
Fig. 3.

Fig. 3. From: Bat3 promotes the membrane integration of tail-anchored proteins.

Bat3 is required for Sec61β-OPG membrane integration. Samples of rabbit reticulocyte lysate were immunodepleted of Bat3 or TRC40, subjected to a parallel control immunodepletion using appropriate chicken or rabbit antibodies, or left completely untreated, as indicated. (A) Upper panel, the ability of different reticulocyte lysate preparations to support the membrane integration of Sec61β-OPG was assessed via N-glycosylation (+gly and −gly) following immunoblotting with the anti-opsin monoclonal antibody. Lower panels, reticulocyte lysate was left untreated or treated with increasing amounts of a chicken anti-Bat3 IgY or a control chicken IgY as indicated, and samples immunodepleted using a secondary antibody. The resulting levels of Bat3, TRC40, Hsp/Hsc70 and SRP54 were determined by immunoblotting as indicated. Individual boxes show scans from one exposure of a single piece of film. (B) Upper panel, the ability of different lysate preparations to support the membrane integration of Sec61β-OPG was determined as described in A. This panel shows the scan from a single piece of film with two irrelevant lanes separating the TRC40 depletion (lane 2) and the parallel control sample (lane 3) removed for simplicity, as indicated by the vertical line. Lower panel, the levels of TRC40 and Bat3 were determined as for A. (C) The ability of different lysate preparations to support the membrane integration of Cytb5-OPG was determined as for A. A small amount of SDS-resistant Cytb5-OPG dimer was observed. In some cases, samples subjected to immunodepletion contained residual immunoglobulin heavy chain (labelled IgG) that crossreacted with the secondary antibody (see A and B, TRC40 immunoblot, white dots). A minor species observed with the TRC40 serum is also indicated (B and C, TRC40 panel, filled square).

Pawel Leznicki, et al. J Cell Sci. 2010 July 1;123(13):2170-2178.

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