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Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport.

Hirata T, Fujita M, Nakamura S, Gotoh K, Motooka D, Murakami Y, Maeda Y, Kinoshita T - Mol. Biol. Cell (2015)

Bottom Line: In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport.Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins.In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan.

No MeSH data available.


Potential role for TMEM87A and TMEM87B in endosome-to-TGN retrograde transport. (A) Overexpression of TMEM87A (TM87A) and TMEM87B (TM87B) partly rescues the delayed transport of proteins in V54KO cells. Transport assay of VFG-GPI (left) or FVG-TM (right) in V54KO cells transiently transfected with the indicated genes. (B) Overexpression of GPR107 and GPR108 does not rescue delayed transport in V54KO cells. Transport assay of VFG-GPI (left) or FVG-TM (right) in V54KO cells transfected with the indicated genes. (C) Subcellular localization of TMEM87A. Wild-type cells were transfected with nontagged or HA-tagged TMEM87A and double-stained with anti-TMEM87A (nontagged version) or anti-HA7 and anti-GPP130, a Golgi marker. Green, TMEM87A; red, GPP130 as a Golgi marker. Scale bars, 10 μm. (D) CTxB retrograde transport at 0 and 60 min. Green, CTxB; red, golgin97. Scale bars, 10 μm. Similar results were obtained in two independent experiments. (E) Quantitative data were obtained as described in the legend to Figure 5C. Data are the means of 10 independent images. Error bars represent SEM (n = 10). Similar results were obtained in two independent experiments.
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Figure 6: Potential role for TMEM87A and TMEM87B in endosome-to-TGN retrograde transport. (A) Overexpression of TMEM87A (TM87A) and TMEM87B (TM87B) partly rescues the delayed transport of proteins in V54KO cells. Transport assay of VFG-GPI (left) or FVG-TM (right) in V54KO cells transiently transfected with the indicated genes. (B) Overexpression of GPR107 and GPR108 does not rescue delayed transport in V54KO cells. Transport assay of VFG-GPI (left) or FVG-TM (right) in V54KO cells transfected with the indicated genes. (C) Subcellular localization of TMEM87A. Wild-type cells were transfected with nontagged or HA-tagged TMEM87A and double-stained with anti-TMEM87A (nontagged version) or anti-HA7 and anti-GPP130, a Golgi marker. Green, TMEM87A; red, GPP130 as a Golgi marker. Scale bars, 10 μm. (D) CTxB retrograde transport at 0 and 60 min. Green, CTxB; red, golgin97. Scale bars, 10 μm. Similar results were obtained in two independent experiments. (E) Quantitative data were obtained as described in the legend to Figure 5C. Data are the means of 10 independent images. Error bars represent SEM (n = 10). Similar results were obtained in two independent experiments.

Mentions: We reasoned that overexpression of a protein involved in VAMP4-mediated endosome-to-TGN retrograde transport would rescue the V54KO transport defect. Therefore we next tried to isolate suppressor genes that, when overexpressed, rescued the transport delay in V54KO cells. A cDNA library derived from human brain was introduced into V54KO cells, and the cDNAs in the transport-restored cell populations were sequenced. From this screening, we obtained TMEM87A, a multipass transmembrane protein belonging to the LU7TM family (from Uniprot; Supplemental Table S3). Overexpression of TMEM87A partially restored the defect in anterograde transport of both VFG-GPI and FVG-TM in V54KO cells (Figure 6A; geometric means of cell surface levels of VFG-GPI and FVG-TM were 1.92 ± 0.22 and 4.45 ± 1.1 times, respectively, those in V54KO+Vec; n = 3). Overexpression of TMEM87B, a close homologue of TMEM87A and a member of the same family, also restored the defect in V54KO cells (Figure 6A; geometric means of cell surface levels of VFG-GPI and FVG-TM were 1.85 ± 0.22 and 3.42 ± 0.99 times, respectively, those in V54KO+Vec; n = 3), suggesting its functional redundancy with TMEM87A. Recently a role for GPR107—another LU7TM family member—was shown in retrograde transport of toxins such as exotoxin A of Pseudomonas aeruginosa and ricin (Carette et al., 2011a; Elling et al., 2011; Tafesse et al., 2014; Zhou et al., 2014). Unlike TMEM87A and TMEM87B, overexpression of GPR107 or GPR108, a member of LU7TM family closely related to GPR107, did not restore the delayed transport of either VFG-GPI or FVG-TM in V54KO cells (Figure 6B; geometric means of cell surface levels of VFG-GPI and FVG-TM in V54KO+GPR107 were 1.21 ± 0.38 and 0.98 ± 0.11 times, respectively, those in V54KO+Vec [n = 2], and those in V54KO+GPR108 were 1.23 ± 0.14 and 1.15 ± 0.027 times, respectively, those in V54KO+Vec [n = 2]). These results suggested the specific function of TMEM87A and TMEM87B. Because these two proteins have not been characterized, we first investigated their localization in mammalian cells. Because endogenous TMEM87A protein could not be detected by immunoblotting and immunofluorescence analysis in HEK293 cells, expression plasmids of N-terminally hemagglutinin (HA)-tagged or C-terminally 3×HA-tagged TMEM87A and TMEM87B were constructed and their functions confirmed based on their ability to restore the V54KO phenotype. Overexpression of N- or C-terminally tagged TMEM87A restored the impairment of anterograde transport of VFG-GPI to an extent similar to that of the nontagged version (Supplemental Figure S3A), indicating that both HA-tagged TMEM87As were functional; by contrast, neither of the tagged TMEM87Bs was functional. Nontagged or N- or C-terminally HA-tagged TMEM87A was transiently expressed in wild-type cells, and the localization of the proteins was analyzed by confocal microscopy. As shown in Figure 6C, nontagged or N- or C-terminally HA-tagged TMEM87A predominantly colocalized with GPP130, indicating its Golgi localization.


Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport.

Hirata T, Fujita M, Nakamura S, Gotoh K, Motooka D, Murakami Y, Maeda Y, Kinoshita T - Mol. Biol. Cell (2015)

Potential role for TMEM87A and TMEM87B in endosome-to-TGN retrograde transport. (A) Overexpression of TMEM87A (TM87A) and TMEM87B (TM87B) partly rescues the delayed transport of proteins in V54KO cells. Transport assay of VFG-GPI (left) or FVG-TM (right) in V54KO cells transiently transfected with the indicated genes. (B) Overexpression of GPR107 and GPR108 does not rescue delayed transport in V54KO cells. Transport assay of VFG-GPI (left) or FVG-TM (right) in V54KO cells transfected with the indicated genes. (C) Subcellular localization of TMEM87A. Wild-type cells were transfected with nontagged or HA-tagged TMEM87A and double-stained with anti-TMEM87A (nontagged version) or anti-HA7 and anti-GPP130, a Golgi marker. Green, TMEM87A; red, GPP130 as a Golgi marker. Scale bars, 10 μm. (D) CTxB retrograde transport at 0 and 60 min. Green, CTxB; red, golgin97. Scale bars, 10 μm. Similar results were obtained in two independent experiments. (E) Quantitative data were obtained as described in the legend to Figure 5C. Data are the means of 10 independent images. Error bars represent SEM (n = 10). Similar results were obtained in two independent experiments.
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Figure 6: Potential role for TMEM87A and TMEM87B in endosome-to-TGN retrograde transport. (A) Overexpression of TMEM87A (TM87A) and TMEM87B (TM87B) partly rescues the delayed transport of proteins in V54KO cells. Transport assay of VFG-GPI (left) or FVG-TM (right) in V54KO cells transiently transfected with the indicated genes. (B) Overexpression of GPR107 and GPR108 does not rescue delayed transport in V54KO cells. Transport assay of VFG-GPI (left) or FVG-TM (right) in V54KO cells transfected with the indicated genes. (C) Subcellular localization of TMEM87A. Wild-type cells were transfected with nontagged or HA-tagged TMEM87A and double-stained with anti-TMEM87A (nontagged version) or anti-HA7 and anti-GPP130, a Golgi marker. Green, TMEM87A; red, GPP130 as a Golgi marker. Scale bars, 10 μm. (D) CTxB retrograde transport at 0 and 60 min. Green, CTxB; red, golgin97. Scale bars, 10 μm. Similar results were obtained in two independent experiments. (E) Quantitative data were obtained as described in the legend to Figure 5C. Data are the means of 10 independent images. Error bars represent SEM (n = 10). Similar results were obtained in two independent experiments.
Mentions: We reasoned that overexpression of a protein involved in VAMP4-mediated endosome-to-TGN retrograde transport would rescue the V54KO transport defect. Therefore we next tried to isolate suppressor genes that, when overexpressed, rescued the transport delay in V54KO cells. A cDNA library derived from human brain was introduced into V54KO cells, and the cDNAs in the transport-restored cell populations were sequenced. From this screening, we obtained TMEM87A, a multipass transmembrane protein belonging to the LU7TM family (from Uniprot; Supplemental Table S3). Overexpression of TMEM87A partially restored the defect in anterograde transport of both VFG-GPI and FVG-TM in V54KO cells (Figure 6A; geometric means of cell surface levels of VFG-GPI and FVG-TM were 1.92 ± 0.22 and 4.45 ± 1.1 times, respectively, those in V54KO+Vec; n = 3). Overexpression of TMEM87B, a close homologue of TMEM87A and a member of the same family, also restored the defect in V54KO cells (Figure 6A; geometric means of cell surface levels of VFG-GPI and FVG-TM were 1.85 ± 0.22 and 3.42 ± 0.99 times, respectively, those in V54KO+Vec; n = 3), suggesting its functional redundancy with TMEM87A. Recently a role for GPR107—another LU7TM family member—was shown in retrograde transport of toxins such as exotoxin A of Pseudomonas aeruginosa and ricin (Carette et al., 2011a; Elling et al., 2011; Tafesse et al., 2014; Zhou et al., 2014). Unlike TMEM87A and TMEM87B, overexpression of GPR107 or GPR108, a member of LU7TM family closely related to GPR107, did not restore the delayed transport of either VFG-GPI or FVG-TM in V54KO cells (Figure 6B; geometric means of cell surface levels of VFG-GPI and FVG-TM in V54KO+GPR107 were 1.21 ± 0.38 and 0.98 ± 0.11 times, respectively, those in V54KO+Vec [n = 2], and those in V54KO+GPR108 were 1.23 ± 0.14 and 1.15 ± 0.027 times, respectively, those in V54KO+Vec [n = 2]). These results suggested the specific function of TMEM87A and TMEM87B. Because these two proteins have not been characterized, we first investigated their localization in mammalian cells. Because endogenous TMEM87A protein could not be detected by immunoblotting and immunofluorescence analysis in HEK293 cells, expression plasmids of N-terminally hemagglutinin (HA)-tagged or C-terminally 3×HA-tagged TMEM87A and TMEM87B were constructed and their functions confirmed based on their ability to restore the V54KO phenotype. Overexpression of N- or C-terminally tagged TMEM87A restored the impairment of anterograde transport of VFG-GPI to an extent similar to that of the nontagged version (Supplemental Figure S3A), indicating that both HA-tagged TMEM87As were functional; by contrast, neither of the tagged TMEM87Bs was functional. Nontagged or N- or C-terminally HA-tagged TMEM87A was transiently expressed in wild-type cells, and the localization of the proteins was analyzed by confocal microscopy. As shown in Figure 6C, nontagged or N- or C-terminally HA-tagged TMEM87A predominantly colocalized with GPP130, indicating its Golgi localization.

Bottom Line: In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport.Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins.In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan.

No MeSH data available.