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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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Identification of novel mammalian TRAPP components. (A) HEK293T cells were transfected with an empty vector (vector) or transfected with either TAP-C2 or TAP-C2L. Lysates were subjected to a two-step affinity purification and fractionated by SDS–PAGE. Bands were excised and subjected to mass spectrometric identification. In some cases, bands were not resolved and the entire eluate was analyzed by mass spectrometry. The bait band refers to either C2 (lane 2) or C2L (lane 3). TTC15/TRAPPC12 is marked with an asterisk since it was not identified in a gel slice but rather following mass spectrometric analysis of a non-gel-resolved protein preparation. Note that the uneven staining of the gel is due to the use of a discontinuous gradient in the resolving portion of the gel. (B) Lysates of HEK293T cells transfected with FLAG-C2L and myc-C2 (lane 1) were treated with preimmune rabbit serum (lane 2) or anti–FLAG IgG (lane 3), and the precipitates were subjected to Western analysis using anti–myc IgG. (C) Eluates following TAP purification were fractionated from cells transfected with an empty plasmid (vector) or with the TAP-C4orf41. The gel was transferred to a PVDF membrane and probed for the presence of the indicated TRAPP proteins. (D) HEK293T cells were transfected with HA-C4orf41. Lysates were incubated with preimmune serum (lane 2), anti-HA (lane 3), or anti-TTC15/C12 (lane 4). Immunoprecipitates were then fractionated by SDS–PAGE and probed for the presence of C2, C2L, C3, C12 or HA (indicating the presence of C4orf41/C11). Inputs representing 10% of the sample precipitated are shown in lane 1.
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Figure 1: Identification of novel mammalian TRAPP components. (A) HEK293T cells were transfected with an empty vector (vector) or transfected with either TAP-C2 or TAP-C2L. Lysates were subjected to a two-step affinity purification and fractionated by SDS–PAGE. Bands were excised and subjected to mass spectrometric identification. In some cases, bands were not resolved and the entire eluate was analyzed by mass spectrometry. The bait band refers to either C2 (lane 2) or C2L (lane 3). TTC15/TRAPPC12 is marked with an asterisk since it was not identified in a gel slice but rather following mass spectrometric analysis of a non-gel-resolved protein preparation. Note that the uneven staining of the gel is due to the use of a discontinuous gradient in the resolving portion of the gel. (B) Lysates of HEK293T cells transfected with FLAG-C2L and myc-C2 (lane 1) were treated with preimmune rabbit serum (lane 2) or anti–FLAG IgG (lane 3), and the precipitates were subjected to Western analysis using anti–myc IgG. (C) Eluates following TAP purification were fractionated from cells transfected with an empty plasmid (vector) or with the TAP-C4orf41. The gel was transferred to a PVDF membrane and probed for the presence of the indicated TRAPP proteins. (D) HEK293T cells were transfected with HA-C4orf41. Lysates were incubated with preimmune serum (lane 2), anti-HA (lane 3), or anti-TTC15/C12 (lane 4). Immunoprecipitates were then fractionated by SDS–PAGE and probed for the presence of C2, C2L, C3, C12 or HA (indicating the presence of C4orf41/C11). Inputs representing 10% of the sample precipitated are shown in lane 1.

Mentions: The yeast orthologue of C2L, Tca17p, was shown to associate loosely with TRAPP and implied, based in part on yeast genetic interactions, to have a TRAPP II–specific function (Montpetit and Conibear, 2009; Scrivens et al., 2009). In an effort to discern a functional difference between C2 and C2L, we precipitated tandem affinity purification (TAP)-tagged C2 and C2L followed by SDS–PAGE and mass spectrometry. Somewhat surprisingly, the pattern of associated bands detected by silver staining was essentially indistinguishable between the two proteins, suggesting that the two proteins are found in equivalent complexes (Figure 1A). The presence of C2 and C2L in the same complex was indeed confirmed as seen by the coprecipitation of myc-tagged C2 with FLAG-tagged C2L (Figure 1B), consistent with the notion that C2L completes the symmetry of the TRAPP core, residing opposite C2, as previously suggested (Scrivens et al., 2009).


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)

Identification of novel mammalian TRAPP components. (A) HEK293T cells were transfected with an empty vector (vector) or transfected with either TAP-C2 or TAP-C2L. Lysates were subjected to a two-step affinity purification and fractionated by SDS–PAGE. Bands were excised and subjected to mass spectrometric identification. In some cases, bands were not resolved and the entire eluate was analyzed by mass spectrometry. The bait band refers to either C2 (lane 2) or C2L (lane 3). TTC15/TRAPPC12 is marked with an asterisk since it was not identified in a gel slice but rather following mass spectrometric analysis of a non-gel-resolved protein preparation. Note that the uneven staining of the gel is due to the use of a discontinuous gradient in the resolving portion of the gel. (B) Lysates of HEK293T cells transfected with FLAG-C2L and myc-C2 (lane 1) were treated with preimmune rabbit serum (lane 2) or anti–FLAG IgG (lane 3), and the precipitates were subjected to Western analysis using anti–myc IgG. (C) Eluates following TAP purification were fractionated from cells transfected with an empty plasmid (vector) or with the TAP-C4orf41. The gel was transferred to a PVDF membrane and probed for the presence of the indicated TRAPP proteins. (D) HEK293T cells were transfected with HA-C4orf41. Lysates were incubated with preimmune serum (lane 2), anti-HA (lane 3), or anti-TTC15/C12 (lane 4). Immunoprecipitates were then fractionated by SDS–PAGE and probed for the presence of C2, C2L, C3, C12 or HA (indicating the presence of C4orf41/C11). Inputs representing 10% of the sample precipitated are shown in lane 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113772&req=5

Figure 1: Identification of novel mammalian TRAPP components. (A) HEK293T cells were transfected with an empty vector (vector) or transfected with either TAP-C2 or TAP-C2L. Lysates were subjected to a two-step affinity purification and fractionated by SDS–PAGE. Bands were excised and subjected to mass spectrometric identification. In some cases, bands were not resolved and the entire eluate was analyzed by mass spectrometry. The bait band refers to either C2 (lane 2) or C2L (lane 3). TTC15/TRAPPC12 is marked with an asterisk since it was not identified in a gel slice but rather following mass spectrometric analysis of a non-gel-resolved protein preparation. Note that the uneven staining of the gel is due to the use of a discontinuous gradient in the resolving portion of the gel. (B) Lysates of HEK293T cells transfected with FLAG-C2L and myc-C2 (lane 1) were treated with preimmune rabbit serum (lane 2) or anti–FLAG IgG (lane 3), and the precipitates were subjected to Western analysis using anti–myc IgG. (C) Eluates following TAP purification were fractionated from cells transfected with an empty plasmid (vector) or with the TAP-C4orf41. The gel was transferred to a PVDF membrane and probed for the presence of the indicated TRAPP proteins. (D) HEK293T cells were transfected with HA-C4orf41. Lysates were incubated with preimmune serum (lane 2), anti-HA (lane 3), or anti-TTC15/C12 (lane 4). Immunoprecipitates were then fractionated by SDS–PAGE and probed for the presence of C2, C2L, C3, C12 or HA (indicating the presence of C4orf41/C11). Inputs representing 10% of the sample precipitated are shown in lane 1.
Mentions: The yeast orthologue of C2L, Tca17p, was shown to associate loosely with TRAPP and implied, based in part on yeast genetic interactions, to have a TRAPP II–specific function (Montpetit and Conibear, 2009; Scrivens et al., 2009). In an effort to discern a functional difference between C2 and C2L, we precipitated tandem affinity purification (TAP)-tagged C2 and C2L followed by SDS–PAGE and mass spectrometry. Somewhat surprisingly, the pattern of associated bands detected by silver staining was essentially indistinguishable between the two proteins, suggesting that the two proteins are found in equivalent complexes (Figure 1A). The presence of C2 and C2L in the same complex was indeed confirmed as seen by the coprecipitation of myc-tagged C2 with FLAG-tagged C2L (Figure 1B), consistent with the notion that C2L completes the symmetry of the TRAPP core, residing opposite C2, as previously suggested (Scrivens et al., 2009).

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