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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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Depletion of either TRAPPC11 or TRAPPC12 perturbs ts045-VSV-G-GFP trafficking. HeLa cells were treated with nonspecific siRNA (A) or siRNA against either C11 (B and D) or C12 (C and E). After 48 h, cells were transfected with a plasmid encoding ts045-VSV-G-GFP. Cells were then shifted to 39.5°C for 6 h (A, left) and subsequently incubated at 32°C for 30 min (all other panels). Cells were then stained with anti-Sec31 or anti-ERGIC53, as indicated. The right panels in (B–E) represent merged images from the two panels directly to their left. The scale bar in (A) is 10 μm, and all other bars are 2 μm.
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Figure 5: Depletion of either TRAPPC11 or TRAPPC12 perturbs ts045-VSV-G-GFP trafficking. HeLa cells were treated with nonspecific siRNA (A) or siRNA against either C11 (B and D) or C12 (C and E). After 48 h, cells were transfected with a plasmid encoding ts045-VSV-G-GFP. Cells were then shifted to 39.5°C for 6 h (A, left) and subsequently incubated at 32°C for 30 min (all other panels). Cells were then stained with anti-Sec31 or anti-ERGIC53, as indicated. The right panels in (B–E) represent merged images from the two panels directly to their left. The scale bar in (A) is 10 μm, and all other bars are 2 μm.

Mentions: As components of TRAPP, C11 and C12 are likely involved in anterograde traffic between the ER and the Golgi. The fragmentation of the Golgi observed upon knockdown is indicative of such a role, but to address this notion directly, we examined the effect of C11 and C12 depletion on the trafficking of the temperature-sensitive, fluorophore-tagged VSV-G mutant ts045-VSV-G-GFP. At temperatures above 38°C, this mutant protein is retained in the ER, but upon shifting to the permissive temperature of 32°C, it is efficiently released, first to the ERGIC and then further through the secretory pathway (Bergmann and Singer, 1983; Scales et al., 1997). HeLa cells were depleted of C11 or C12 using siRNA for 48 h and then transfected with a plasmid containing ts045-VSV-G-GFP (VSV-G). The following day, VSV-G was blocked in the ER by incubation at the restrictive temperature for 6 h. VSV-G was then released by shifting to 32°C for 30 min in the presence of cycloheximide. In cells treated with a nonspecific oligonucleotide, the fluorescent signal appeared in the perinuclear region within 30 min and colocalized with the Golgi marker Man II (Figure 5A). On knockdown of C11 or C12, however, the mutant protein did not traffic to a perinuclear region typical of the Golgi, but rather accumulated in punctate structures (Figure 5, B–E, and Supplemental Figure 7). Confocal microscopy demonstrated that these punctae costain for ERGIC53 (Figure 5, D and E) and are closely apposed to Sec31 (Figure 5, B and C), markers for the ERGIC and ER-exit sites, respectively (Figure 5). These results are consistent with the VSV-G visualized here, representing either cargo at an ER exit site or peripheral ERGIC apposed to an ER exit site, similar to that reported by Mironov et al., 2003.


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)

Depletion of either TRAPPC11 or TRAPPC12 perturbs ts045-VSV-G-GFP trafficking. HeLa cells were treated with nonspecific siRNA (A) or siRNA against either C11 (B and D) or C12 (C and E). After 48 h, cells were transfected with a plasmid encoding ts045-VSV-G-GFP. Cells were then shifted to 39.5°C for 6 h (A, left) and subsequently incubated at 32°C for 30 min (all other panels). Cells were then stained with anti-Sec31 or anti-ERGIC53, as indicated. The right panels in (B–E) represent merged images from the two panels directly to their left. The scale bar in (A) is 10 μm, and all other bars are 2 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113772&req=5

Figure 5: Depletion of either TRAPPC11 or TRAPPC12 perturbs ts045-VSV-G-GFP trafficking. HeLa cells were treated with nonspecific siRNA (A) or siRNA against either C11 (B and D) or C12 (C and E). After 48 h, cells were transfected with a plasmid encoding ts045-VSV-G-GFP. Cells were then shifted to 39.5°C for 6 h (A, left) and subsequently incubated at 32°C for 30 min (all other panels). Cells were then stained with anti-Sec31 or anti-ERGIC53, as indicated. The right panels in (B–E) represent merged images from the two panels directly to their left. The scale bar in (A) is 10 μm, and all other bars are 2 μm.
Mentions: As components of TRAPP, C11 and C12 are likely involved in anterograde traffic between the ER and the Golgi. The fragmentation of the Golgi observed upon knockdown is indicative of such a role, but to address this notion directly, we examined the effect of C11 and C12 depletion on the trafficking of the temperature-sensitive, fluorophore-tagged VSV-G mutant ts045-VSV-G-GFP. At temperatures above 38°C, this mutant protein is retained in the ER, but upon shifting to the permissive temperature of 32°C, it is efficiently released, first to the ERGIC and then further through the secretory pathway (Bergmann and Singer, 1983; Scales et al., 1997). HeLa cells were depleted of C11 or C12 using siRNA for 48 h and then transfected with a plasmid containing ts045-VSV-G-GFP (VSV-G). The following day, VSV-G was blocked in the ER by incubation at the restrictive temperature for 6 h. VSV-G was then released by shifting to 32°C for 30 min in the presence of cycloheximide. In cells treated with a nonspecific oligonucleotide, the fluorescent signal appeared in the perinuclear region within 30 min and colocalized with the Golgi marker Man II (Figure 5A). On knockdown of C11 or C12, however, the mutant protein did not traffic to a perinuclear region typical of the Golgi, but rather accumulated in punctate structures (Figure 5, B–E, and Supplemental Figure 7). Confocal microscopy demonstrated that these punctae costain for ERGIC53 (Figure 5, D and E) and are closely apposed to Sec31 (Figure 5, B and C), markers for the ERGIC and ER-exit sites, respectively (Figure 5). These results are consistent with the VSV-G visualized here, representing either cargo at an ER exit site or peripheral ERGIC apposed to an ER exit site, similar to that reported by Mironov et al., 2003.

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