Protein N-glycosylation is one of the fundamental metabolic pathways in cell fate. Deciphering how N-glycosylation controls specific metabolic and signaling events has become important to unraveling the underlying basis of cellular behavior. In yeasts, outer chain branching is initiated in the Golgi apparatus by the α-1,6-mannosyltransferase Och1p. Here, we reported that altered mitochondrial functionality and oxidative stress take place in K. lactis cells carrying a mutation in KlOCH1 gene. The mutant cells showed also a reduction in the calcium content and in the expression of genes related to calcium signalling.
Although a direct link between N-glycosylation defects and mitochondrial functionality has not been reported, underglycosylation of proteins destined for the mitochondria could interfere with, or abolish, the import of these proteins into the organelle. Indeed, a 45 kDa N-glycoprotein has been identified in rat liver inner mitochondrial membranes that physically interacts with complex I and the F1F0-ATP synthase ; Tim11p, its yeast homologue, has one potential N-glycosylation site. Also, mutations in the signal recognition particle (SRP) receptor have been shown to disrupt the reticular structure of both the ER and mitochondria in yeast , suggesting that a proper ER structure and/or functionality is required for maintaining the mitochondrial network. It is conceivable that the underglycosylation of proteins has adverse effects on the early secretory compartments structure and functionality, which, in turn, influences mitochondria characteristics.
Moreover, we found that KlCMD1 gene was a suppressor in Kloch1-1 cells of either ROS accumulation or sensitivity to the oxidative stress, when H2O2 was added in the growth medium. On the other hand, the increased dosage of this Ca2+-signalling gene was not able to increase the survival rate of the mutant cells undergoing a treatment with cytotoxic concentration of the same oxidant agent. These data suggest that, in the mutant cells, the altered defence mechanisms against high concentration of a ROS generator were not fixed by calmodulin itself.
The Kloch1-1 cells also showed a reduction in the calcium content, accompanied by a reduction in the expression of the KlCMD1, KlCNA1 and KlCNB1 genes, encoding for key components of the calmodulin/calcineurin signalling pathway. In fungi, conserved signal transduction pathways control fundamental aspects of growth, development and reproduction. Two important classes of fungal signalling pathways are the mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway. They are triggered by an array of stimuli and target a broad range of downstream effectors such as transcription factors, cytoskeletal proteins, protein kinases and other enzymes, thereby regulating processes such reproduction, morphogenesis and stress response [19, 20]. We can thus hypothesize a possible activation of a MAPK signaling in Kloch1-1 cells that in turn could down modulate the calcium signaling pathway. It has been found that in K. lactis cells deleted in PMR1 gene and sharing phenotypes with the Kloch1-1 mutant, such as cell wall defects, oxidative stress and altered calcium homeostasis, the HOG1 MAPK cascade resulted activated .
S. cerevisiae calmodulin, a Ca2+ binding protein, regulates many cell processes both depending or not upon the intervention of Ca2+ ions. Among those Ca2+-dependent is the organization of the actin cytoskeleton; moreover mutations in CMD1 resulted colethal, suggestive of functional interactions, with the inactivation of genes encoding components of the glycosylation pathways like ANP1, CWH8 and MNN10 ; mutations in such genes also result in altered morphology of actin cytoskeleton. We should also take into account that, although och1 deletion mutant of S.cerevisae in the BY4741 background was sensitive to EGTA, the mutant strain was not altered in the expression of calmodulin and calcineurin genes (Zanni et al., unpublished results). However, we can not exclude the possibility that a reduction in the activity of the calcium signalling proteins can occur in the OCH1 deleted cells.
Mitochondrial plasticity and functionality strongly depend upon the interactions between mitochondria and cytoskeleton. Several shape-related proteins have been described in S.cerevisiae, localized on the mitochondria surface and reported to interact with actin [23, 24]; however the individual role and underlaying mechanisms are still unsolved.
Another unanswered question is how do cells change the mitochondrial shape upon cell signals. In the case of calcium signalling, a relevant player could well be Gem1p, a member of the Miro GTPase family ; Gem1p is also localized on the outer mitochondrial membrane with its GTPase domain and, most notably, its EF-hand calcium binding domain exposed in the cytosol.
We are tempting to speculate that the altered calcium availability we observed in Kloch1-1 cells could be originated by a defective calcium membrane channel Mid1/Cch1. In fact, it has been demonstrated that S. cerevisiae Mid1 requires a full glycosylation to correctly localize and assemble at the level of the plasma membrane .
In mammalian cells Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 and dephosphorylation at serine 637, both calcineurin-dependent, are associated with increase in Drp1 translocation to mitochondria [27, 28].
Nevertheless, one cannot exclude that defective KlOCH1 gene induce reduced glycosylation of other proteins relevant for calcium handling; scrutiny of such picture will deserve future work.