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Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation.

ABSTRACT: Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca(2+) exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca(2+) uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca(2+)/H(+) antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn(2+) to the cells.

SUBMITTER: Colinet AS

PROVIDER: S-EPMC4830978 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMost secretory proteins contain signal peptides that direct their sorting to the ER and secreted via the conventional ER/Golgi transport pathway, while some signal-peptide-lacking proteins have been shown to export through ER/Golgi independent secretory pathways. Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that is active against both prokaryotic and eukaryotic cells. The hygromycin phosphotransferase (HYG(R)) can phosphorylate and inactivate the hygromycin B, and has been widely used as a positive selective marker in the construction of transgenic plants. However, the localization and trafficking of HYG(R) in plant cells remain unknown. Synaptotagmins (SYTs) are involved in controlling vesicle endocytosis and exocytosis as calcium sensors in animal cells, while their functions in plant cells are largely unclear.Methodology/principal findingsWe found Arabidopsis synaptotagmin SYT2 was localized on the Golgi apparatus by immunofluorescence and immunogold labeling. Surprisingly, co-expression of SYT2 and HYG(R) caused hypersensitivity of the transgenic Arabidopsis plants to hygromycin B. HYG(R), which lacks a signal sequence, was present in the cytoplasm as well as in the extracellular space in HYG(R)-GFP transgenic Arabidopsis plants and its secretion is not sensitive to brefeldin A treatment, suggesting it is not secreted via the conventional secretory pathway. Furthermore, we found that HYG(R)-GFP was truncated at carboxyl terminus of HYG(R) shortly after its synthesis, and the cells deficient SYT2 failed to efficiently truncate HYG(R)-GFP,resulting in HYG(R)-GFP accumulated in prevacuoles/vacuoles, indicating that SYT2 was involved in HYG(R)-GFP trafficking and secretion.Conclusion/significanceThese findings reveal for the first time that SYT2 is localized on the Golgi apparatus and regulates HYG(R)-GFP secretion via the unconventional protein transport from the cytosol to the extracelluar matrix in plant cells.

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Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation.

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