Fission Yeast Study Reveals How Gdt1, Gdt2 and Pmr1 Team Up to Balance Calcium and Manganese

Calcium and manganese may be humble minerals, but inside every eukaryotic cell they are tightly guarded commodities. The enzymes that decorate newly made proteins with sugar chains, the machinery that sorts cargo through the secretory pathway, and the walls that give cells their shape all depend on precise concentrations of these two divalent cations within the endoplasmic reticulum and Golgi apparatus. A new study published in International Microbiology by Farman Ullah and Ying Huang of Nanjing Normal University now provides the first detailed functional portrait of how the fission yeast Schizosaccharomyces pombe manages this balancing act, and the results point to a three-protein partnership with an unexpected division of labor.

The central characters of the study belong to the UPF0016 family, an evolutionarily ancient group of membrane proteins found across fungi, plants, algae, and animals. In budding yeast, the family member Gdt1 works alongside the P-type ATPase Pmr1, a calcium and manganese pump, to maintain ion levels in the Golgi lumen. In humans, the corresponding protein TMEM165 is so essential that mutations in its gene cause congenital disorders of glycosylation, a group of serious developmental conditions. Yet S. pombe is unusual: unlike most species examined so far, it carries two UPF0016 paralogs, Gdt1 and Gdt2, whose physiological roles had remained essentially unexplored.

To dissect those roles, the researchers constructed single-gene deletion mutants lacking gdt1, gdt2, or pmr1, as well as all three possible double mutants, using one-step gene replacement with antibiotic resistance markers. They then subjected the strains to a battery of stress tests, spotting serial dilutions of cells onto agar plates laced with calcium chloride at 300, 400, and 750 millimolar, or manganese chloride at 0.8, 1.5, and 2 millimolar. The pattern that emerged was striking. Cells lacking gdt1 were markedly hypersensitive to both cations, while deletion of gdt2 or pmr1 alone produced only mild growth defects under the tested conditions.

The double mutants told a more dramatic story. Combining gdt1 and gdt2 deletions produced the most severe calcium-sensitive phenotype, with virtually no growth at 750 millimolar CaCl2, evidence that the two paralogs partially substitute for one another. The gdt1 pmr1 double mutant displayed strong synthetic sickness, its growth nearly undetectable at 400 millimolar calcium even though each single mutant survived with moderate defects. The gdt2 pmr1 combination was weaker but still明显 impaired, with residual growth persisting even at the highest dose. Together these genetic interactions indicate that Gdt1, Gdt2, and Pmr1 operate in complementary, overlapping pathways rather than as a single linear chain.

A chelation experiment confirmed the mechanism behind the toxicity. When the calcium-specific chelator EGTA was added to plates containing 400 millimolar CaCl2, growth of both the gdt1 mutant and the gdt2 pmr1 double mutant was substantially restored. This rescue demonstrates that the growth failures stem specifically from an inability to cope with excess free extracellular calcium, not from a general fitness defect. Manganese assays echoed the calcium results: the gdt1 gdt2 double mutant was nearly unable to grow at 2 millimolar MnCl2, while the gdt2 pmr1 strain grew reasonably well even at that concentration, showing that Gdt1 alone can sustain manganese tolerance across the range tested.

Growth curves in liquid culture reinforced the hierarchy. Wild-type cells reached an optical density of about 13.2 after 32 hours, whereas the gdt1 mutant plateaued at only 5.3, the lowest of the single mutants, followed by gdt2 at 7.4 and pmr1 at 8.5. Every double mutant fared worse than any single mutant, with the gdt1 pmr1 combination most severely affected at an optical density near 3.1. The data identify Gdt1 as the primary growth determinant among the three proteins and reinforce the conclusion that its function cannot be fully replaced by its paralog or by the ATPase.

Microscopy added a visible dimension to the genetics. Under standard conditions, healthy fission yeast cells are elongated rods, but all mutant strains were significantly shorter and rounder, averaging around 7.0 to 7.2 micrometers in length compared with 11.7 micrometers for wild type. Stress amplified these defects in distinctive ways: high calcium drove abnormal elongation, with the gdt1 pmr1 double mutant stretching to an average of 15.7 micrometers, while manganese produced the most extreme swelling, widening mutant cells to as much as 8.2 micrometers, nearly double the wild-type diameter. These distortions are consistent with compromised cell wall biosynthesis and disrupted glycosylation, processes known to depend on luminal calcium and manganese in the secretory pathway.

The study also delved into structure. Multiple sequence alignments across UPF0016 homologs from budding yeast, Candida albicans, Arabidopsis, Chlamydomonas, and humans showed that the two fission yeast proteins share 48.44 percent amino acid identity and both retain the family’s signature motifs, EIGDKT and EWGDRS, whose conserved glutamate and aspartate residues had previously been shown to be essential for calcium transport. Topology predictions, however, revealed a divergence: SpGdt1 is predicted to span the membrane five times, while SpGdt2 carries six transmembrane helices. In silico modeling with the MIB2 server mapped candidate metal-binding residues, identifying Glu38 and Glu190 as likely manganese coordinators in Gdt1, supported by Asp41 and Ser43 for the larger calcium ion, and Glu61 and Glu214 in the corresponding positions of Gdt2.

Phylogenetic analysis placed the fission yeast proteins on their own branch, separate from the single homolog of budding yeast, suggesting that the gdt1 and gdt2 pair arose from a lineage-specific gene duplication after the two yeast lineages split. Localization predictions using DeepLoc 2.0, together with curated PomBase and ORFeome annotations, assign SpGdt1 primarily to the endoplasmic reticulum and SpGdt2 to both the ER and Golgi, a compartmental pattern that differs from the Golgi residency of the budding yeast protein and may explain why the fission yeast pump Pmr1 itself localizes mainly to ER membranes in earlier fluorescence studies.

The implications reach well beyond a single yeast species. Because glycosylation enzymes require manganese and calcium cofactors, defects in UPF0016-dependent ion regulation can cascade into failures of protein maturation, secretion, and cell wall construction, which is precisely why human TMEM165 mutations manifest as congenital glycosylation disorders and why plant homologs such as PAM71 are indispensable for chloroplast manganese uptake and efficient photosynthesis. By establishing that Gdt1 is the dominant determinant of divalent cation tolerance in fission yeast while Gdt2 and Pmr1 provide complementary support, the study completes a missing evolutionary link and sets the stage for direct transport assays, motif-directed mutants, and organelle-level imaging to confirm how these conserved transporters move metal ions across secretory pathway membranes.

Subject of Research: Regulation of calcium and manganese ion homeostasis by UPF0016 family proteins Gdt1, Gdt2 and the P-type ATPase Pmr1 in the fission yeast Schizosaccharomyces pombe

Article Title: Gdt1, Gdt2 and the P-type ATPase Pmr1 regulate divalent cations (Ca²⁺ and Mn²⁺) in the fission yeast Schizosaccharomyces pombe

Article References: Ullah, F., & Huang, Y. (2026). Gdt1, Gdt2 and the P-type ATPase Pmr1 regulate divalent cations (Ca²⁺ and Mn²⁺) in the fission yeast Schizosaccharomyces pombe. International Microbiology. https://doi.org/10.1007/s10123-026-00887-0

Image Credits: AI Generated

DOI: 10.1007/s10123-026-00887-0

Keywords: Schizosaccharomyces pombe, UPF0016 family, Gdt1, Gdt2, Pmr1, calcium homeostasis, manganese transport, Golgi apparatus, endoplasmic reticulum, protein glycosylation, cell wall integrity, TMEM165