TMEM165 deficiencies lead to one of the Congenital Disorders of Glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from Golgi manganese homeostasis defect and that Mn2+ supplementation was sufficient to rescue a normal glycosylation. In this paper we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn2+ concentrations, TMEM165 was degraded into lysosomes. The Mn2+ induced lysosomal targeting of TMEM165 occurred via a Rab7 and Rab5 independent pathways, suggesting a direct trafficking from the Golgi to lysosomes in response to Mn2+. Remarkably, the variant p.E108G recently identified in a novel TMEM165-CDG patient, was found insensitive to Mn2+ supplementation. Moreover, this mutation abolished the function of TMEM165, suggesting that a transport function may be necessary for its regulation.
Altogether our results identified the Golgi protein TMEM165 as a novel cytosolic Mn2+ sensor in mammalian cells and pointed to the crucial importance of the cytosolical ELGDK motif in both Mn2+ sensitivity and function.
The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca/Mn homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca. The use of Pmr1p mutants either defective for Ca or Mn transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn inside the Golgi lumen when Pmr1p exclusively transports Ca. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn/Catransport.
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