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. 2011;6(8):e23350.
doi: 10.1371/journal.pone.0023350. Epub 2011 Aug 15.

The adaptor function of TRAPPC2 in mammalian TRAPPs explains TRAPPC2-associated SEDT and TRAPPC9-associated congenital intellectual disability

Min Zong  1 , Xing-gang Wu, Cecilia W L Chan, Mei Y Choi, Hsiao Chang Chan, Julian A Tanner, Sidney Yu
Affiliations

The adaptor function of TRAPPC2 in mammalian TRAPPs explains TRAPPC2-associated SEDT and TRAPPC9-associated congenital intellectual disability

Min Zong et al. PLoS One. 2011.

Abstract

Background: The TRAPP (Transport protein particle) complex is a conserved protein complex functioning at various steps in vesicle transport. Although yeast has three functionally and structurally distinct forms, TRAPPI, II and III, emerging evidence suggests that mammalian TRAPP complex may be different. Mutations in the TRAPP complex subunit 2 (TRAPPC2) cause X-linked spondyloepiphyseal dysplasia tarda, while mutations in the TRAPP complex subunit 9 (TRAPPC9) cause postnatal mental retardation with microcephaly. The structural interplay between these subunits found in mammalian equivalent of TRAPPI and those specific to TRAPPII and TRAPPIII remains largely unknown and we undertook the present study to examine the interaction between these subunits. Here, we reveal that the mammalian equivalent of the TRAPPII complex is structurally distinct from the yeast counterpart thus leading to insight into mechanism of disease.

Principal findings: We analyzed how TRAPPII- or TRAPPIII- specific subunits interact with the six-subunit core complex of TRAPP by co-immunoprecipitation in mammalian cells. TRAPPC2 binds to TRAPPII-specific subunit TRAPPC9, which in turn binds to TRAPPC10. Unexpectedly, TRAPPC2 can also bind to the putative TRAPPIII-specific subunit, TRAPPC8. Endogenous TRAPPC9-positive TRAPPII complex does not contain TRAPPC8, suggesting that TRAPPC2 binds to either TRAPPC9 or TRAPPC8 during the formation of the mammalian equivalents of TRAPPII or TRAPPIII, respectively. Therefore, TRAPPC2 serves as an adaptor for the formation of these complexes. A disease-causing mutation of TRAPPC2, D47Y, failed to interact with either TRAPPC9 or TRAPPC8, suggesting that aspartate 47 in TRAPPC2 is at or near the site of interaction with TRAPPC9 or TRAPPC8, mediating the formation of TRAPPII and/or TRAPPIII. Furthermore, disease-causing deletional mutants of TRAPPC9 all failed to interact with TRAPPC2 and TRAPPC10.

Conclusions: TRAPPC2 serves as an adaptor for the formation of TRAPPII or TRAPPIII in mammalian cells. The mammalian equivalent of TRAPPII is likely different from the yeast TRAPPII structurally.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Interactions between putative TRAPPII specific subunits and TRAPPI core subunits.
A. GFP-tagged TRAPPC10 was co-transfected with the indicated Myc-tagged TRAPPI core subunits in COS cells for co-IP with antibody against c-Myc. The presence of pulled down TRAPPC10 was detected by immunoblotting with anti-GFP antibody (top panel). The amount of precipitated Myc-tagged proteins is shown in second panel from the top. The levels of protein expression in the transfected lysates are shown in bottom two panels. B. GFP-tagged TRAPPC9 and indicated Myc-tagged TRAPPI core subunits were co-transfected into COS cells. Co-IP and subsequent detection by immunoblotting was performed as above. The experiments shown are representatives of three independent experiments.
Figure 2
Figure 2. TRAPPC8 interacts mainly with TRAPPC2.
A. Increasing amounts of GFP-TRAPPC8 cDNA were co-transfected with a constant amount of Myc-TRAPPC2 and Myc-TRAPPC9. The levels of protein expression of all three TRAPP subunits were determined by immunoblotting the lysates of the transfected cells. B. Increasing amounts of GFP-TRAPPC9 cDNA were co-transfected with a constant amount of Myc-TRAPPC2 and Myc-TRAPPC8. C. GFP-tagged TRAPPC8 and the indicated Myc-tagged TRAPPI core subunits were co-transfected into COS cells. Co-IP and subsequent detection by immunoblotting was performed as in Figure 1. The experiments shown are representatives of three independent experiments.
Figure 3
Figure 3. Native TRAPP complex containing TRAPPC9 does not contain TRAPPC8.
Native TRAPP complex was isolated by IP using antibody against TRAPPC9 (lane 3). The presence of TRAPPC9 (top panel), TRAPPC2 (bottom panel) and TRAPPC8 (middle panel) was detected by immunoblotting using antibodies specific to these proteins. IP using antibody against lysosomal marker LAMP2 serves as negative control (lane 2). HEK293 lysate used in this experiment (approximately 1% of input) is shown in lane 1. The experiment shown is a representative of two independent experiments.
Figure 4
Figure 4. TRAPPC2(D47Y) mutant shows a reduced binding capacity for TRAPPC9 and TRAPPC8.
A. Lysates from co-transfections of GFP-TRAPPC9 or -TRAPPC8 with either Myc-TRAPPC2 or -TRAPPC2(D47Y) were subjected to IP using anti-Myc antibody. Wildtype Myc-TRAPPC2 was able to pull down GFP-TRAPPC9 (lane 1) and GFP-TRAPPC8 (lane 3). D47Y mutant, however, showed drastically reduced ability to pull down GFP-TRAPPC9 (lane 2), and failed to pull down GFP-TRAPPC8 (lane 4). The expression levels of these proteins are shown in lanes 5–8. B. A quantification of the degree of binding was performed using data collected from three experiments similar to the one shown in A. The intensity of the TRAPPC8 or TRAPPC9 co-precipitated by D47Y is expressed as a percentage of the wildtype TRAPPC2 binding in each co-IP experiment. Error bars = S.E.M. C. COS cells transfected with wildtype or D47Y Myc-tagged TRAPPC2 were stained with antibodies against Golgin-97 and AF488-secondary antibody (green signal, left panels). Myc-TRAPPC2 expression was detected with TRITC-conjugated 9E10 antibody (red signals, left panels). Golgi fragmentation was quantified by counting cells with fragmented Golgin-97 signals (right panel). In three independent experiments, the total number of cells counted is 352 (non-transfected), 277 (wildtype) and 323 (D47Y). Error bar = S.E.M. D. CHO cells transfected with wildtype or D47Y Myc-tagged TRAPPC2 were stained with antibodies against TRAPPC9 and AF488-secondary antibody (top left panels). The expression of Myc-tagged TRAPPC2 was detected by TRITC-9E10 (red) (bottom left panels). The ability of wildtype or D47Y to disrupt the native localization of TRAPPC9 was quantified using images obtained similarly to those shown in the top panels (right panel). A total of 119, 98 and 149 cells were counted in D47Y, wildtype TRAPPC2, and non-transfected CHO cells, respectively.
Figure 5
Figure 5. Disease-causing deletional mutants of TRAPPC9 fail to bind to TRAPPC2 or TRAPPC10.
A. GFP -GAP273, -TRAPPC9, -TRAPPC9(L772Δ) and -TRAPPC9(R475X) were tested for the interaction with Myc-tagged TRAPPC2. The levels of protein expression in the transfected lysates are shown in lanes 1 to 4. Lanes 5 to 8 are immunoprecipitants. Full-length TRAPPC9 was efficiently pulled down by Myc-TRAPPC2 (lane 6), but not the deletion mutants of TRAPPC9 (lane 7 and 8), or negative control GFP-GAP273 (lane 5). B. Full-length and deletional mutants of TRAPPC9 were tested for the interaction with Myc-tagged TRAPPC10. The levels of protein expression in the transfected lysates are shown in lanes 1 to 4. Lanes 5 to 8 are immunoprecipitants. Full-length TRAPPC9 was efficiently pulled down by Myc-TRAPPC10 (lane 6), but not the deletional mutants (lane 7 and 8), or negative control GFP-GAP273 (lane 5). The experiments shown are representatives of two independent experiments.
Figure 6
Figure 6. Schematic diagram of the yeast and mammalian TRAPP complexes.
A. Schematic diagram of yeast TRAPP complexes. B. Schematic diagram of the putative mammalian TRAPP complexes. The subunits that interact with TRAPPII- or TRAPPIII- specific subunits are labeled in yellow.
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