N -Glycosylation of MRS2 balances aerobic and anaerobic energy production by reducing rapid mitochondrial Mg ²⁺ influx in conditions of high glucose or impaired respiratory chain function

N- linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. While N- glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial protein N- linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, is N- glycosylated to various extents depending on cellular bioenergetic status. Both N -glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A or Lens culinaris agglutinin. Following peptide: N- glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher M _r MRS2 bands gel-shifted to lower M _r and loss of lectin affinity was seen. BRL 3A cells treated with two different N- linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-L-norleucine, resulted in decreased intensity or loss of the higher M _r MRS2 isoform. To investigate the possible functional role of MRS2 N- glycosylation, we measured rapid Mg ²⁺ influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-L-norleucine. Interestingly, rapid Mg ²⁺ influx capacity increased in mitochondria isolated from BRL 3A cells treated with either N- glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media or the glycolytic inhibitor 2-deoxyglucose or by minimizing glucose concentration similarly reduced the N- glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg ²⁺ influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction of N- glycosylated MRS2, with decreased rapid Mg ²⁺ influx capacity. Collectively, these data provide strong evidence that MRS2 N -glycosylation is directly involved in the regulation of mitochondrial matrix Mg ²⁺ , dynamically communicating relative cellular nutrient status and bioenergetic capacity by serving as a physiologic brake on the influx of mitochondrial matrix Mg ²⁺ under conditions of glucose excess or mitochondrial bioenergetic impairment.