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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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Size exclusion chromatography of TRAPP components. (A) Lysates from HEK293T cells left untransfected or transfected with V5-C8 or V5-C10 were fractionated on a Superdex 200 size exclusion column. Fractions (0.5 ml) were collected, and every second fraction was analyzed by Western analysis using antibodies against endogenous C2, C2L, C3, C11, and C12, or anti-V5 to detect transfected C8 or C10. (B) HeLa cells were treated with a nonspecific siRNA (KD: NS) or siRNA against C11 (KD: C11). Lysates were fractionated by size exclusion chromatography as above and subjected to Western analysis using anti–C2 antibody (top two panels) or anti–C12 antibody (bottom two panels). (C) HeLa cells transfected with HA-C11 were treated with a nonspecific siRNA (NS) or siRNA against C11 or C12. Equal amounts of the lysates were analyzed by Western blotting and probed for the presence of tubulin (loading control), C2, C2L, C3, C11 (using anti-HA), and C12.
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Figure 2: Size exclusion chromatography of TRAPP components. (A) Lysates from HEK293T cells left untransfected or transfected with V5-C8 or V5-C10 were fractionated on a Superdex 200 size exclusion column. Fractions (0.5 ml) were collected, and every second fraction was analyzed by Western analysis using antibodies against endogenous C2, C2L, C3, C11, and C12, or anti-V5 to detect transfected C8 or C10. (B) HeLa cells were treated with a nonspecific siRNA (KD: NS) or siRNA against C11 (KD: C11). Lysates were fractionated by size exclusion chromatography as above and subjected to Western analysis using anti–C2 antibody (top two panels) or anti–C12 antibody (bottom two panels). (C) HeLa cells transfected with HA-C11 were treated with a nonspecific siRNA (NS) or siRNA against C11 or C12. Equal amounts of the lysates were analyzed by Western blotting and probed for the presence of tubulin (loading control), C2, C2L, C3, C11 (using anti-HA), and C12.

Mentions: We next examined the fractionation of the newly identified proteins by size exclusion chromatography. Consistent with our previous results regarding TRAPP organization in mammalian cells (Scrivens et al., 2009), V5-tagged C8 and endogenous C11 and C12 eluted in the same high-molecular-weight fraction as other TRAPP subunits examined, including endogenous C2, C2L, C3, and V5-tagged C10 (Figure 2A). A second peak for V5-C10 was seen and is most likely due to ectopic expression because it caused a shift in the low-molecular-weight pool of C2L (Supplemental Figure 4) and was not seen for the endogenous C10 protein (Yamasaki et al., 2009). Curiously, endogenous C12 was observed to elute in the same high-molecular-weight pool as TRAPP, as well as a 400- to 500-kDa fraction distinct from other TRAPP components. The components of this smaller C12 assembly are currently being characterized.


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)

Size exclusion chromatography of TRAPP components. (A) Lysates from HEK293T cells left untransfected or transfected with V5-C8 or V5-C10 were fractionated on a Superdex 200 size exclusion column. Fractions (0.5 ml) were collected, and every second fraction was analyzed by Western analysis using antibodies against endogenous C2, C2L, C3, C11, and C12, or anti-V5 to detect transfected C8 or C10. (B) HeLa cells were treated with a nonspecific siRNA (KD: NS) or siRNA against C11 (KD: C11). Lysates were fractionated by size exclusion chromatography as above and subjected to Western analysis using anti–C2 antibody (top two panels) or anti–C12 antibody (bottom two panels). (C) HeLa cells transfected with HA-C11 were treated with a nonspecific siRNA (NS) or siRNA against C11 or C12. Equal amounts of the lysates were analyzed by Western blotting and probed for the presence of tubulin (loading control), C2, C2L, C3, C11 (using anti-HA), and C12.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Size exclusion chromatography of TRAPP components. (A) Lysates from HEK293T cells left untransfected or transfected with V5-C8 or V5-C10 were fractionated on a Superdex 200 size exclusion column. Fractions (0.5 ml) were collected, and every second fraction was analyzed by Western analysis using antibodies against endogenous C2, C2L, C3, C11, and C12, or anti-V5 to detect transfected C8 or C10. (B) HeLa cells were treated with a nonspecific siRNA (KD: NS) or siRNA against C11 (KD: C11). Lysates were fractionated by size exclusion chromatography as above and subjected to Western analysis using anti–C2 antibody (top two panels) or anti–C12 antibody (bottom two panels). (C) HeLa cells transfected with HA-C11 were treated with a nonspecific siRNA (NS) or siRNA against C11 or C12. Equal amounts of the lysates were analyzed by Western blotting and probed for the presence of tubulin (loading control), C2, C2L, C3, C11 (using anti-HA), and C12.
Mentions: We next examined the fractionation of the newly identified proteins by size exclusion chromatography. Consistent with our previous results regarding TRAPP organization in mammalian cells (Scrivens et al., 2009), V5-tagged C8 and endogenous C11 and C12 eluted in the same high-molecular-weight fraction as other TRAPP subunits examined, including endogenous C2, C2L, C3, and V5-tagged C10 (Figure 2A). A second peak for V5-C10 was seen and is most likely due to ectopic expression because it caused a shift in the low-molecular-weight pool of C2L (Supplemental Figure 4) and was not seen for the endogenous C10 protein (Yamasaki et al., 2009). Curiously, endogenous C12 was observed to elute in the same high-molecular-weight pool as TRAPP, as well as a 400- to 500-kDa fraction distinct from other TRAPP components. The components of this smaller C12 assembly are currently being characterized.

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