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C4orf41 and TTC-15 are mammalian TRAPP components with a role at an early stage in ER-to-Golgi trafficking.

Scrivens PJ, Noueihed B, Shahrzad N, Hul S, Brunet S, Sacher M - Mol. Biol. Cell (2011)

Bottom Line: Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage.We further present a binary interaction map for all known mammalian TRAPP components and evidence that TRAPP oligomerizes.Our data are consistent with the absence of a TRAPP I-equivalent complex in mammalian cells, suggesting that the fundamental unit of mammalian TRAPP is distinct from that characterized in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Concordia University, Montreal, Quebec, Canada.

ABSTRACT
TRAPP is a multisubunit tethering complex implicated in multiple vesicle trafficking steps in Saccharomyces cerevisiae and conserved throughout eukarya, including humans. Here we confirm the role of TRAPPC2L as a stable component of mammalian TRAPP and report the identification of four novel components of the complex: C4orf41, TTC-15, KIAA1012, and Bet3L. Two of the components, KIAA1012 and Bet3L, are mammalian homologues of Trs85p and Bet3p, respectively. The remaining two novel TRAPP components, C4orf41 and TTC-15, have no homologues in S. cerevisiae. With this work, human homologues of all the S. cerevisiae TRAPP proteins, with the exception of the Saccharomycotina-specific subunit Trs65p, have now been reported. Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage. We further present a binary interaction map for all known mammalian TRAPP components and evidence that TRAPP oligomerizes. Our data are consistent with the absence of a TRAPP I-equivalent complex in mammalian cells, suggesting that the fundamental unit of mammalian TRAPP is distinct from that characterized in S. cerevisiae.

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Mammalian TRAPP forms oligomers. (A) HEK293T cells were cotransfected with FLAG-C2L/myc-C2L, V5-C10/GFP-C10, V5-C11/HA-C11, or V5-C12/GFP-C12. Lysates were treated with preimmune rabbit serum (lane 1), anti–FLAG (for the C2L transfections), or anti–V5 (for the C10, C11, and C12 transfections) IgG and then probed with anti–myc (for the C2L transfection), anti–GFP (for the C10 and C12 transfections), or anti–HA (for the C11 transfection) IgG. (B) The lysates from (A) were fractionated by gel filtration chromatography. The high-molecular-weight TRAPP-containing fractions were pooled and immunoprecipitated and probed as in (A). (C) A model for the architecture of mammalian TRAPP built from yeast two-hybrid and coimmunoprecipitation data. Subunits shaded in blue are arranged based on the previously published architecture of the subcomplex (Kim et al., 2006). The high-molecular-weight subunits (C8–C12) are represented by a single mauve oval. Given interactions between high–molecular-weight components with proteins at both ends of the TRAPP “core,” a network of high-molecular-weight subunit interactions among themselves, and oligomerization of the complex as indicated in (A) and (B), two TRAPP “cores” could be bound via high-molecular-weight subunit interactions in trans. The two models differ with respect to the orientation of the second “core.” See the text for details.
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Figure 7: Mammalian TRAPP forms oligomers. (A) HEK293T cells were cotransfected with FLAG-C2L/myc-C2L, V5-C10/GFP-C10, V5-C11/HA-C11, or V5-C12/GFP-C12. Lysates were treated with preimmune rabbit serum (lane 1), anti–FLAG (for the C2L transfections), or anti–V5 (for the C10, C11, and C12 transfections) IgG and then probed with anti–myc (for the C2L transfection), anti–GFP (for the C10 and C12 transfections), or anti–HA (for the C11 transfection) IgG. (B) The lysates from (A) were fractionated by gel filtration chromatography. The high-molecular-weight TRAPP-containing fractions were pooled and immunoprecipitated and probed as in (A). (C) A model for the architecture of mammalian TRAPP built from yeast two-hybrid and coimmunoprecipitation data. Subunits shaded in blue are arranged based on the previously published architecture of the subcomplex (Kim et al., 2006). The high-molecular-weight subunits (C8–C12) are represented by a single mauve oval. Given interactions between high–molecular-weight components with proteins at both ends of the TRAPP “core,” a network of high-molecular-weight subunit interactions among themselves, and oligomerization of the complex as indicated in (A) and (B), two TRAPP “cores” could be bound via high-molecular-weight subunit interactions in trans. The two models differ with respect to the orientation of the second “core.” See the text for details.

Mentions: For the yeast two-hybrid analysis, each TRAPP subunit was tested in both the bait and prey vectors against all subunits listed in Table 1. All previously identified interactions based on earlier structural work with some of the mammalian subunits (Kim et al., 2006) were recapitulated in our extensive binary interaction map, suggesting that this is a valid means to assess interacting partners within this complex (Table 3). The strongest interactions were seen between C2/C2L/C6 and the high-molecular-weight proteins C10, C11, and C12. In addition, the high-molecular-weight proteins appear to have extensive and strong interactions among themselves. These data support a model of mammalian TRAPP in which the high-molecular-weight proteins associate with each other and with one or both ends of the complex via interactions with C6, C2L, and/or C2 (see Figure 7C).


C4orf41 and TTC-15 are mammalian TRAPP components with a role at an early stage in ER-to-Golgi trafficking.

Scrivens PJ, Noueihed B, Shahrzad N, Hul S, Brunet S, Sacher M - Mol. Biol. Cell (2011)

Mammalian TRAPP forms oligomers. (A) HEK293T cells were cotransfected with FLAG-C2L/myc-C2L, V5-C10/GFP-C10, V5-C11/HA-C11, or V5-C12/GFP-C12. Lysates were treated with preimmune rabbit serum (lane 1), anti–FLAG (for the C2L transfections), or anti–V5 (for the C10, C11, and C12 transfections) IgG and then probed with anti–myc (for the C2L transfection), anti–GFP (for the C10 and C12 transfections), or anti–HA (for the C11 transfection) IgG. (B) The lysates from (A) were fractionated by gel filtration chromatography. The high-molecular-weight TRAPP-containing fractions were pooled and immunoprecipitated and probed as in (A). (C) A model for the architecture of mammalian TRAPP built from yeast two-hybrid and coimmunoprecipitation data. Subunits shaded in blue are arranged based on the previously published architecture of the subcomplex (Kim et al., 2006). The high-molecular-weight subunits (C8–C12) are represented by a single mauve oval. Given interactions between high–molecular-weight components with proteins at both ends of the TRAPP “core,” a network of high-molecular-weight subunit interactions among themselves, and oligomerization of the complex as indicated in (A) and (B), two TRAPP “cores” could be bound via high-molecular-weight subunit interactions in trans. The two models differ with respect to the orientation of the second “core.” See the text for details.
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Related In: Results  -  Collection

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Figure 7: Mammalian TRAPP forms oligomers. (A) HEK293T cells were cotransfected with FLAG-C2L/myc-C2L, V5-C10/GFP-C10, V5-C11/HA-C11, or V5-C12/GFP-C12. Lysates were treated with preimmune rabbit serum (lane 1), anti–FLAG (for the C2L transfections), or anti–V5 (for the C10, C11, and C12 transfections) IgG and then probed with anti–myc (for the C2L transfection), anti–GFP (for the C10 and C12 transfections), or anti–HA (for the C11 transfection) IgG. (B) The lysates from (A) were fractionated by gel filtration chromatography. The high-molecular-weight TRAPP-containing fractions were pooled and immunoprecipitated and probed as in (A). (C) A model for the architecture of mammalian TRAPP built from yeast two-hybrid and coimmunoprecipitation data. Subunits shaded in blue are arranged based on the previously published architecture of the subcomplex (Kim et al., 2006). The high-molecular-weight subunits (C8–C12) are represented by a single mauve oval. Given interactions between high–molecular-weight components with proteins at both ends of the TRAPP “core,” a network of high-molecular-weight subunit interactions among themselves, and oligomerization of the complex as indicated in (A) and (B), two TRAPP “cores” could be bound via high-molecular-weight subunit interactions in trans. The two models differ with respect to the orientation of the second “core.” See the text for details.
Mentions: For the yeast two-hybrid analysis, each TRAPP subunit was tested in both the bait and prey vectors against all subunits listed in Table 1. All previously identified interactions based on earlier structural work with some of the mammalian subunits (Kim et al., 2006) were recapitulated in our extensive binary interaction map, suggesting that this is a valid means to assess interacting partners within this complex (Table 3). The strongest interactions were seen between C2/C2L/C6 and the high-molecular-weight proteins C10, C11, and C12. In addition, the high-molecular-weight proteins appear to have extensive and strong interactions among themselves. These data support a model of mammalian TRAPP in which the high-molecular-weight proteins associate with each other and with one or both ends of the complex via interactions with C6, C2L, and/or C2 (see Figure 7C).

Bottom Line: Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage.We further present a binary interaction map for all known mammalian TRAPP components and evidence that TRAPP oligomerizes.Our data are consistent with the absence of a TRAPP I-equivalent complex in mammalian cells, suggesting that the fundamental unit of mammalian TRAPP is distinct from that characterized in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Concordia University, Montreal, Quebec, Canada.

ABSTRACT
TRAPP is a multisubunit tethering complex implicated in multiple vesicle trafficking steps in Saccharomyces cerevisiae and conserved throughout eukarya, including humans. Here we confirm the role of TRAPPC2L as a stable component of mammalian TRAPP and report the identification of four novel components of the complex: C4orf41, TTC-15, KIAA1012, and Bet3L. Two of the components, KIAA1012 and Bet3L, are mammalian homologues of Trs85p and Bet3p, respectively. The remaining two novel TRAPP components, C4orf41 and TTC-15, have no homologues in S. cerevisiae. With this work, human homologues of all the S. cerevisiae TRAPP proteins, with the exception of the Saccharomycotina-specific subunit Trs65p, have now been reported. Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage. We further present a binary interaction map for all known mammalian TRAPP components and evidence that TRAPP oligomerizes. Our data are consistent with the absence of a TRAPP I-equivalent complex in mammalian cells, suggesting that the fundamental unit of mammalian TRAPP is distinct from that characterized in S. cerevisiae.

Show MeSH
Related in: MedlinePlus