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The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity.

Hwang GW, Murai Y, Takahashi T, Naganuma A - Sci Rep (2014)

Bottom Line: We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury.Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles.The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.

ABSTRACT
Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

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Effect of simultaneous deletion of two genes involved in different parts of intracellular protein trafficking on the sensitivity of yeast cells to methylmercury.(a, b, c) Yeast cells (106 cells/ml) with deletions of the indicated gene(s) were grown in SD liquid media with or without methylmercuric chloride at the indicated concentrations. After incubation for 3 hrs at 30°C, cells from each strain were diluted in sterilised water to 107 cells/ml. Five microlitres of each of the resulting cell suspensions were spotted on agar-solidified SD media. For further details, refer to the Figure 1c legend.
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f3: Effect of simultaneous deletion of two genes involved in different parts of intracellular protein trafficking on the sensitivity of yeast cells to methylmercury.(a, b, c) Yeast cells (106 cells/ml) with deletions of the indicated gene(s) were grown in SD liquid media with or without methylmercuric chloride at the indicated concentrations. After incubation for 3 hrs at 30°C, cells from each strain were diluted in sterilised water to 107 cells/ml. Five microlitres of each of the resulting cell suspensions were spotted on agar-solidified SD media. For further details, refer to the Figure 1c legend.

Mentions: There are two types of proteins transported to vacuoles via endosomes; one becomes functional in the vacuole, and the other is degraded in the vacuole12. Membrane proteins such as signalling receptors and transporters are incorporated into the cell by endocytosis and then transported to the vacuole via the endosome and degraded, resulting in regulation of their cell surface abundance1213. Newly synthesised membrane proteins in the cell are transported to the trans-Golgi network (TGN) from the endoplasmic reticulum via the Golgi body. They are subsequently sorted in the TGN. Some proteins are transported to the cell membrane to become functional there, and the rest are transported to the vacuole via the endosome and degraded. Six pathways are involved in protein transportation to vacuoles: 1) a transportation pathway from the endoplasmic reticulum to the TGN, 2) a direct transportation pathway from the TGN to vacuoles (the alkaline phosphatase (ALP) pathway), 3) a transportation pathway from the TGN to endosomes, 4) the uptake of proteins into endosomes (the multivesicular body (MVB) sorting pathway), 5) endocytosis, and 6) endosome-vacuole fusion1213 (Figure 2a). Our study showed that proteins involved in the transportation pathway from the TGN to endosomes (Vps45 and Vps68) and those involved in the MVB sorting pathway (Snf8, Vps4, Vps28, and Vta1) play a role in the enhancement of methylmercury toxicity. However, because this study used a simple screening method conducted in the presence of a fixed concentration of methylmercury (60 nM), some factors involved in the protein transportation pathway from the TGN to vacuoles may not be detected despite potentially impacting methylmercury toxicity. To elucidate the relationship between methylmercury toxicity and proteins involved in the protein transportation pathway from the TGN to vacuoles and to identify the pathway playing a role in the enhancement of methylmercury toxicity among those transportation pathways, we investigated the sensitivity of each gene deletion mutant to proteins known to be involved in those pathways. Several single-gene deletion mutants have a similar level of sensitivity to methylmercury as do wild-type yeast. These include, the genes encoding Apl5 and Apl6, both of which are involved in the pathway for direct transportation of substrate proteins from the TGN to vacuoles without passing through endosomes (the second pathway, as previously listed) and the genes encoding End3, Ent1, Ent2, and Ent4, all of which are involved in endocytosis (the fifth pathway, as previously listed; data not shown). Based on these results, it appears that neither of these pathways is involved in methylmercury toxicity enhancement. A subset of single-gene deletion mutants showed higher resistance to methylmercury compared to wild-type yeast. These included genes encoding Vps45 and Pep12, both of which are involved in transportation from the TGN to endosomes (the third pathway, as previously listed), genes encoding Vps27, Hse1, Stp22, Srn2, Vps28, Vps36, Vps25, Snf8, Did4, Vps20, Vps24, Snf7, and Doa4, all of which are involved in the MVB sorting pathway (the fourth pathway, as previously listed), and genes encoding Vam3 and Vam7, both of which are involved in endosome-vacuole fusion (the sixth pathway previously listed; Figure 2b). We also examined the methylmercury sensitivity of yeast cells with genetic deletion of Ypt7 and Mon1, which are involved in the same pathway as Vam3 and Vam7. However, these deletion mutants did not affect the yeast's sensitivity to methylmercury. It has been reported that the knockout of Ypt7 or Mon1 inhibited approximately 50% of vacuole transport activity and that the knockout of Vam3 or Vam7 inhibited a majority of vacuole transport activity14. Therefore, Ypt7 and Mon1 may not be essential for vacuole transport pathway via Vam3 and Vam7. From these results, it appears that the protein transportation system from the TGN to vacuoles via endosomes is involved in the enhancement of methylmercury toxicity. However, the three pathways identified in the enhancement of methylmercury toxicity (pathways three, four, and six) may enhance toxicity independently via different mechanisms. Therefore, we prepared double deletion mutants with deletions of Vps45 (involved in transportation from the TGN to endosomes) or Vam7 (involved in endosome-vacuole fusion) in addition to the deletion of the Vps27 gene involved in the MVB sorting pathway (vps45Δvps27Δ and vps27Δ vam7Δ) and studied their sensitivity to methylmercury. In this experiment, we selected Vps27 as the key factor of the MVB sorting pathway because Vps27 is known to function upstream in the MVB sorting pathway. Single-gene deletion mutants with VPS27, VPS45, or VAM7 showed strong resistance to methylmercury. However, an additive increase in resistance level was not observed during double deletion of VPS45 and VPS27 (Figure 3a) or VPS27 and VAM7 (Figure 3b). These results suggest that transportation from the TGN to endosomes (pathway three), the MVB sorting pathway (pathway four), and endosome-vacuole fusion (pathway six), are jointly involved in the enhancement of methylmercury toxicity. Therefore there may be proteins in the cell that enhance methylmercury toxicity via transportation from the TGN to vacuoles via endosomes.


The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity.

Hwang GW, Murai Y, Takahashi T, Naganuma A - Sci Rep (2014)

Effect of simultaneous deletion of two genes involved in different parts of intracellular protein trafficking on the sensitivity of yeast cells to methylmercury.(a, b, c) Yeast cells (106 cells/ml) with deletions of the indicated gene(s) were grown in SD liquid media with or without methylmercuric chloride at the indicated concentrations. After incubation for 3 hrs at 30°C, cells from each strain were diluted in sterilised water to 107 cells/ml. Five microlitres of each of the resulting cell suspensions were spotted on agar-solidified SD media. For further details, refer to the Figure 1c legend.
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Related In: Results  -  Collection

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Show All Figures
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f3: Effect of simultaneous deletion of two genes involved in different parts of intracellular protein trafficking on the sensitivity of yeast cells to methylmercury.(a, b, c) Yeast cells (106 cells/ml) with deletions of the indicated gene(s) were grown in SD liquid media with or without methylmercuric chloride at the indicated concentrations. After incubation for 3 hrs at 30°C, cells from each strain were diluted in sterilised water to 107 cells/ml. Five microlitres of each of the resulting cell suspensions were spotted on agar-solidified SD media. For further details, refer to the Figure 1c legend.
Mentions: There are two types of proteins transported to vacuoles via endosomes; one becomes functional in the vacuole, and the other is degraded in the vacuole12. Membrane proteins such as signalling receptors and transporters are incorporated into the cell by endocytosis and then transported to the vacuole via the endosome and degraded, resulting in regulation of their cell surface abundance1213. Newly synthesised membrane proteins in the cell are transported to the trans-Golgi network (TGN) from the endoplasmic reticulum via the Golgi body. They are subsequently sorted in the TGN. Some proteins are transported to the cell membrane to become functional there, and the rest are transported to the vacuole via the endosome and degraded. Six pathways are involved in protein transportation to vacuoles: 1) a transportation pathway from the endoplasmic reticulum to the TGN, 2) a direct transportation pathway from the TGN to vacuoles (the alkaline phosphatase (ALP) pathway), 3) a transportation pathway from the TGN to endosomes, 4) the uptake of proteins into endosomes (the multivesicular body (MVB) sorting pathway), 5) endocytosis, and 6) endosome-vacuole fusion1213 (Figure 2a). Our study showed that proteins involved in the transportation pathway from the TGN to endosomes (Vps45 and Vps68) and those involved in the MVB sorting pathway (Snf8, Vps4, Vps28, and Vta1) play a role in the enhancement of methylmercury toxicity. However, because this study used a simple screening method conducted in the presence of a fixed concentration of methylmercury (60 nM), some factors involved in the protein transportation pathway from the TGN to vacuoles may not be detected despite potentially impacting methylmercury toxicity. To elucidate the relationship between methylmercury toxicity and proteins involved in the protein transportation pathway from the TGN to vacuoles and to identify the pathway playing a role in the enhancement of methylmercury toxicity among those transportation pathways, we investigated the sensitivity of each gene deletion mutant to proteins known to be involved in those pathways. Several single-gene deletion mutants have a similar level of sensitivity to methylmercury as do wild-type yeast. These include, the genes encoding Apl5 and Apl6, both of which are involved in the pathway for direct transportation of substrate proteins from the TGN to vacuoles without passing through endosomes (the second pathway, as previously listed) and the genes encoding End3, Ent1, Ent2, and Ent4, all of which are involved in endocytosis (the fifth pathway, as previously listed; data not shown). Based on these results, it appears that neither of these pathways is involved in methylmercury toxicity enhancement. A subset of single-gene deletion mutants showed higher resistance to methylmercury compared to wild-type yeast. These included genes encoding Vps45 and Pep12, both of which are involved in transportation from the TGN to endosomes (the third pathway, as previously listed), genes encoding Vps27, Hse1, Stp22, Srn2, Vps28, Vps36, Vps25, Snf8, Did4, Vps20, Vps24, Snf7, and Doa4, all of which are involved in the MVB sorting pathway (the fourth pathway, as previously listed), and genes encoding Vam3 and Vam7, both of which are involved in endosome-vacuole fusion (the sixth pathway previously listed; Figure 2b). We also examined the methylmercury sensitivity of yeast cells with genetic deletion of Ypt7 and Mon1, which are involved in the same pathway as Vam3 and Vam7. However, these deletion mutants did not affect the yeast's sensitivity to methylmercury. It has been reported that the knockout of Ypt7 or Mon1 inhibited approximately 50% of vacuole transport activity and that the knockout of Vam3 or Vam7 inhibited a majority of vacuole transport activity14. Therefore, Ypt7 and Mon1 may not be essential for vacuole transport pathway via Vam3 and Vam7. From these results, it appears that the protein transportation system from the TGN to vacuoles via endosomes is involved in the enhancement of methylmercury toxicity. However, the three pathways identified in the enhancement of methylmercury toxicity (pathways three, four, and six) may enhance toxicity independently via different mechanisms. Therefore, we prepared double deletion mutants with deletions of Vps45 (involved in transportation from the TGN to endosomes) or Vam7 (involved in endosome-vacuole fusion) in addition to the deletion of the Vps27 gene involved in the MVB sorting pathway (vps45Δvps27Δ and vps27Δ vam7Δ) and studied their sensitivity to methylmercury. In this experiment, we selected Vps27 as the key factor of the MVB sorting pathway because Vps27 is known to function upstream in the MVB sorting pathway. Single-gene deletion mutants with VPS27, VPS45, or VAM7 showed strong resistance to methylmercury. However, an additive increase in resistance level was not observed during double deletion of VPS45 and VPS27 (Figure 3a) or VPS27 and VAM7 (Figure 3b). These results suggest that transportation from the TGN to endosomes (pathway three), the MVB sorting pathway (pathway four), and endosome-vacuole fusion (pathway six), are jointly involved in the enhancement of methylmercury toxicity. Therefore there may be proteins in the cell that enhance methylmercury toxicity via transportation from the TGN to vacuoles via endosomes.

Bottom Line: We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury.Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles.The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.

ABSTRACT
Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

Show MeSH
Related in: MedlinePlus