H+ and Pi Byproducts of Glycosylation Affect Ca2+ Homeostasis and Are Retrieved from the Golgi Complex by Homologs of TMEM165 and XPR1

Abstract

Glycosylation reactions in the Golgi complex and the endoplasmic reticulum utilize nucleotide sugars as donors and produce inorganic phosphate (Pi) and acid (H⁺) as byproducts. Here we show that homologs of mammalian XPR1 and TMEM165 (termed Erd1 and Gdt1) recycle luminal Pi and exchange luminal H⁺ for cytoplasmic Ca²⁺, respectively, thereby promoting growth of yeast cells in low Pi and low Ca²⁺ environments. As expected for reversible H⁺/Ca²⁺ exchangers, Gdt1 also promoted growth in high Ca²⁺ environments when the Golgi-localized V-ATPase was operational but had the opposite effect when the V-ATPase was eliminated. Gdt1 activities were negatively regulated by calcineurin signaling and by Erd1, which recycled the Pi byproduct of glycosylation reactions and prevented the loss of this nutrient to the environment via exocytosis. Thus, Erd1 transports Pi in the opposite direction from XPR1 and other EXS family proteins and facilitates byproduct removal from the Golgi complex together with Gdt1.

Keywords: Calcium; Golgi complex; glycosylation; homeostasis; phosphate.

Figure 1

Model of Ca²⁺ homeostasis and Golgi glycosylation, with results from a genome-wide screen. (A) Model of known ion or nucleotide sugar transporters (yellow) in the vacuole and Golgi complex that are studied here along with modes of regulation (blue) by the Crz1 transcription factor, calcineurin, calmodulin, and high cytosolic Ca²⁺. Gdt1 and Erd1 (orange) are putative transporters of the byproducts of glycosylation reactions in the Golgi complex such as H⁺ [produced by mannosyltransferases (MTases)] and Pi [produced by nucleoside triphosphate diphosphatases (NTDPases)] that were identified in genetic screens. (B) Results of a genetic screen for knockout mutants that specifically exhibit hypersensitivity to elevated Ca²⁺ and/or Ca²⁺ plus FK506 in the growth medium. The numbers indicate growth relative to wild-type controls (smaller numbers indicate slower growth) with the strongest effects highlighted (yellow). The 23 filtered mutants were ranked from vcx1∆-like (orange) to pmc1∆-like (blue) based on the difference between the two Ca²⁺ conditions.

Figure 2

Vacuolar V-ATPase determines directionality of Vcx1 operation. (A) Ca²⁺ tolerance assays were performed in duplicate in YPDS medium (black bars) or in the same medium containing 0.2 µg/ml FK506 (gray bars) as described in Materials and Methods, and the derived IC50 values (±SD) for the indicated mutants were plotted on a log scale. (B) The indicated strains were transformed with plasmid pKC190 that bears the PMC1-lacZ reporter gene (Cunningham and Fink 1996) and β-galactosidase activity was measured 4 hr after log-phase cells were shifted to YPDS medium containing 100 mM CaCl₂. Results from three independent transformants were averaged (±SD). * P < 0.05, ** P < 0.01.

Figure 3

Gdt1 promotes Ca²⁺ detoxification independent of Vcx1 and Pmc1. (A) A panel of Saccharomyces cerevisiae strains lacking Gdt1, Vcx1, and Pmc1 in all possible combinations was assayed for Ca²⁺ tolerance in the presence (gray bars) or absence (black bars) of FK506 as described in Figure 2A. (B) A strain expressing epitope-tagged Gdt1-TAP was grown to log phase in YPDS medium containing 200 mM CaCl₂ and/or 1 µg/ml FK506, harvested, lysed, and analyzed by SDS-PAGE and western blotting using anti-TAP polyclonal (top) and anti-tubulin monoclonal (bottom) antibodies.

Figure 4

Gdt1 supplies essential Ca²⁺ independent of Pmr1, and reverse-mode activity of Gdt1 is blocked by Golgi V-ATPase. Mn²⁺ (A) and BAPTA (B and C) tolerance assays were performed in YPD medium, and raw data (A and B) or derived IC50 values (C; ±SD) were plotted on log scales. (D) Expression of PMC1-lacZ reporter gene was measured as described in Figure 2B except the medium contained either 0 mM (white bars) or 50 mM (gray bars) supplemental CaCl₂. * P < 0.05, ** P < 0.01.

Figure 5

Erd1 supplies essential Pi. The indicated mutant strains were washed and inoculated into an SC medium containing various concentrations of Pi. After 24 hr incubation, the concentration of Pi that enabled 50% maximal growth (i.e., ED50) was derived and the averages (±SD) of triplicate measurements were plotted. The erd1∆ mutation increased the ED50 by 1.5-fold, 1.4-fold, and 2.9-fold, respectively, in the wild-type (WT), pho84∆, and pho-∆5 (pho84∆ pho87∆ pho89∆ pho90∆ pho91∆) backgrounds. ** P < 0.01, *** P < 0.001. Note the 15-fold change of scale between pho-∆5 strains (B) and other strains (A).

Figure 6

Erd1 prevents Pi loss to the environment. The indicated mutant strains were grown to log phase in SC medium containing tracer levels of [32]Pi radioisotope, washed extensively in Pi-free SC medium, and then in the same medium (A and B) or in SC medium (C). At the indicated times of incubation at 30°, aliquots were removed and cell-free supernatants were analyzed by liquid scintillation counting (A and C) or by TLC (B). Charts illustrate the averages (±SD) of three biological replicates.

Figure 7

Erd1-sensitive losses of Pi depend on exocytosis and on transport of GDP-mannose into the Golgi complex. (A) Pi losses were measured after 2 hr incubation as in Figure 6A except that sec1-1^ts and sec1-1^ts erd1∆ mutant strains were used at a permissive temperature (30°; black bars) or a non-permissive temperature (37°; gray bars) during the incubation in Pi-free medium. (B) Western blot of Vrg4-AID-FLAG strains after 1 hr exposure to 100 µM auxin in SC medium. (C) Erd1-sensitive losses of Pi (i.e., the difference between erd1∆ and ERD1 pairs of strains) were measured for wild-type and Vrg4-AID-FLAG strain backgrounds that were exposed to 100 µM auxin (gray bars) or not (black bars) starting 1 hr before the shifts to Pi-free medium. *** P < 0.001.