First published online 22 February 2005
doi: 10.1242/jcs.01723
Journal of Cell Science 118, 1209-1222 (2005)
Published by The Company of Biologists 2005
Mammalian Bet3 functions as a cytosolic factor participating in transport from the ER to the Golgi apparatus
Eva Loh1,
Frank Peter2,
V. Nathan Subramaniam3 and
Wanjin Hong1,*
1 Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Republic of Singapore
2 Office of Life Sciences, MD 11 Level 2, #02-04, 10 Medical Drive, Clinical Research Center, Singapore 117597, Republic of Singapore
3 Membrane Transport Laboratory, Cancer and Cell Biology Division, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, QLD 4029, Australia
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Fig. 1. Summary of mammalian TRAPP subunits. (A) Subunits of the TRAPP complex: the name of the subunit, residue number of yeast or human protein, the amino acid (AA) sequence identity between yeast and human protein, and the presence/absence in TRAPP1 and II are indicated. (B) Amino acid sequence alignment of Trs33B of difference species with human Trs33A and yeast Trs33p (h: human; m: mouse; zf: zebrafish). (C) Bet5, TRS20 and TRS23 are related. The alignment of amino acid sequences of human Bet5, TRS20 and TRS23 is shown. (D) Related sequence homology among human Bet3, TRS31, TRS33A and TRS33B. Identical as well as conserved residues are highlighted in gray.
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Fig. 2. Northern blot analysis of Bet3 mRNA in eight mouse tissues. A multiple tissue blot (Clontech) containing 2 µg of polyA+ RNA from the indicated tissues was hybridized with 32P-labeled Bet3 probes and processed for autoradiography.
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Fig. 3. (A) Distribution of Bet3 in various subcellular fractions derived from rat liver. 25 µg of protein from the indicated fractions (PNS, postnuclear supernatant; TM, total membrane; M, microsome-enriched membrane; G, Golgi-enriched membranes; C, cytosol) was separated by SDS-PAGE and stained with Coomassie Blue R-250 (right panel) or transferred to filters and immunoblotted with antibodies against Bet3 (left panel) or GS28 (centre panel). (B) Bet3 is present predominantly in the cytosol. NRK cells were fractionated into total membrane (TM; odd lanes) and cytosol (C; even lanes) fractions. Equivalent fractions of TM and C were separated by SDS-PAGE, transferred to a filter and immunoblotted to detect Bet3 (lanes 1 and 2), GS28 (lanes 3 and 4), ß-actin (lanes 5 and 6) or ß-tubulin (lanes 7 and 8). Their distributions between TM and C fractions were quantitated as shown below the blot. (C) Bet3 is a peripheral membrane protein. Golgi-enriched membranes were extracted with PBS, 1 M KCl, 2.5 M urea, 150 mM sodium carbonate (pH 11.5), or 1% sodium deoxycholate (1% DOC), respectively. The resulting supernatants (S) and pellets (P) were separated by SDS-PAGE, transferred to a filter and immunoblotted to detect GS28 (upper panel) or Bet3 (lower panel).
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Fig. 4. Bet3 is distributed in the cytosol. (A) NRK cells were fixed, permeabilized and double labeled with Bet3 antibodies (a and d) and a monoclonal antibody against ß-COP (b and e). The cytosolic labeling of Bet3 (d) but not ß-COP (e) was abolished by prior incubation of the antibodies with Bet3-His6. The merged images are shown in c and f. Bar, 10 µm. (B) Double labeling of Bet3 antibodies (a and d) and GS28 antibodies (b and e). NRK cells were permeabilized with saponin after (a-c) or before (d-f) fixation with paraformaldehyde. The merged images are shown in c and f. Bar, 10 µm. (C) HeLa cells were transiently transfected to express GFP-Bet3, which displayed cytosolic distribution in live cells expressing low-to-medial levels of GFP-Bet3. Some nuclear accumulation was detected in cells expressing very high levels (marked by white arrow).
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Fig. 5. Antibodies against Bet3 inhibit in vitro transport of VSV-G from the ER to the Golgi apparatus. Semi-intact NRK cells were incubated in the presence of cytosol and ATP on ice (lane 1) or at 32°C for 60 minutes (lanes 2-12). Increasing amounts of anti-Bet3 antibodies were added as indicated (lanes 4-9). Control assays were supplemented with 2 µg of antibody against GS28 (lane 3), 2 µg anti-Bet3 antibodies pre-incubated with 2 µg of GST-Bet3 (lane 10), 2 µg heat-inactivated anti-Bet3 antibodies (lane 11), or 2 µg GST-Bet3 alone (lane 12), respectively. Transport was measured by monitoring the percentage of conversion of the total pool of VSV-G from the endo-H-sensitive to the endo-H-resistant Golgi form (A). The transport was quantified (B).
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Fig. 6. Antibodies against Bet3 functionally inhibit the cytosolic pool of Bet3. Semi-intact NRK cells (lanes 4, 7, 10) were incubated with 2 µg of the indicated antibodies for 1 hour on ice, washed by centrifugation and resuspended in standard transport cocktail. Likewise, cytosol (lanes 5, 8, 11) or combinations of semi-intact cells and cytosol (lanes 3, 6, 9) were incubated with 2 µg of the indicated antibodies for 1 hour on ice. After supplementing with complete transport cocktail, assays were then incubated for 60 minutes on ice (lane 1) or at 32°C (lanes 2 to 11). The extent of VSV-G transport was monitored (A) and quantified (B).
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Fig. 8. Bet3 acts before the EGTA-sensitive step in ER-Golgi transport in vitro. As outlined in (A), permeabilized NRK cells, cytosol and ATP were incubated in the absence (lanes 1 and 2) or presence of 8 mM EGTA (lanes 3-12) on ice (lane 1) or at 32°C (lanes 2-12) for 60 minutes (stage I). The membranes were then collected by a brief spin and resuspended in fresh transport cocktail including fresh ATP and cytosol (lanes 2-11) or Bet3 immunodepleted cytosol (Bet3 drlc, lane 12). Reagents were added at stage I or stage II as indicated in (C). Transport was measured by the conversion of the VSV-G protein to the endo-H-resistant form (B) and quantified (C).
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Fig. 9. Bet3 is functionally required after Sec13 but before Rab1 and  -SNAP. Semi-intact NRK cells were incubated in transport cocktail with the indicated immunodepleted cytosols for 90 minutes at 32°C (stage I). The cells were subsequently washed by centrifugation and resuspended in fresh transport cocktail supplemented with the indicated immunodepleted cytosols for an additional 45 minutes (stage II). The extent of VSV-G transported to the Golgi was quantified (B).
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Fig. 10. Two pools of Bet3 in the cytosol as revealed by gel filtration analysis. (A) Rat liver cytosol (panels a and b) or denatured cytosol (panels c and d) was resolved by gel filtration and the resulting fractions were analyzed by immunoblotting to detect Bet3 (panels a and c) or ß-COP (panels b and d). (B) GST-Bet3 alone (panel a) or GST-Bet3 pre-incubated with Bet3-depleted rlc (panel b) were resolved by gel filtration and the resulting fractions were analyzed by immunoblotting to detect GST-Bet3. Also shown is the distribution of Bet3 in control cytosol (panel c).
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© The Company of Biologists Ltd 2005
