Manganese-induced turnover of TMEM165

Abstract

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 a Golgi manganese homeostasis defect and that Mn²⁺ supplementation was sufficient to rescue normal glycosylation. In the present paper, we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn²⁺ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G, recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether, the present study identified the Golgi protein TMEM165 as a novel Mn²⁺-sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn²⁺-induced degradation of TMEM165.

Keywords: Golgi apparatus; TMEM165; congenital disorders of glycosylation; glycosylation; manganese.

Figure 1. TMEM is rapidly degraded in response to Mn

(A) Steady state cellular level of TMEM165 and GPP130. HeLa cells were treated with MnCl2 500 μM for 0 to 8h. Total cell lysates were prepared, subjected to SDS-PAGE and Western blot with the indicated antibodies. Right panel shows the quantification of TMEM165 and GPP130 protein levels after normalization with actin (Number of experiments (N) = 2; *** = P value < 0,001). (B) Steady state cellular level of TMEM165. HEK293 cells were treated with MnCl2 from 0 to 50 μM for 36h. Total cell lysates were prepared, subjected to SDS-PAGE and Western blot with the indicated antibodies. Lower panel shows the quantification of TMEM165 protein levels after normalization with actin (N = 2; *** = P value < 0,001). (C) Steady state cellular level of GPP130 in the same experimental conditions as described in (B). (D) HeLa cells were incubated with MnCl2 500 μM for 1 and 4h, fixed and labeled with antibodies against TMEM165, GPP130 and GM130 before confocal microscopy visualization. White arrows point to some GPP130 positive vesicles.

Figure 2. Plasma membrane TMEM165 is also degraded by Mn2+ and Ca2+ compete with Mn2+ for TMEM165 degradation

(A) Cell surface biotinylation was performed in absence and presence of MnCl2 500 μM. Samples were prepared as described in materials and methods and subjected to SDS-PAGE and Western blot with the indicated antibodies. Lower panel shows the quantification of TMEM165 protein levels. The plasma membrane panel is separated from the cellular panel as we had to longer expose films to reveal the bands. (B) HEK293 cells were incubated with MnCl2 500 μM and/or CaCl2 2mM for 4h, then subjected to SDS-PAGE and Western blot with the indicated antibodies. Right panel shows the quantification of TMEM165 protein levels after normalization with actin (N = 2; *** = P value < 0,001). (C) HEK293 cells were incubated with MnCl2 500 μM and/or CaCl2 2mM for 4h then fixed and labeled with antibodies against TMEM165 and GM130 before confocal microscopy visualization (N = 2; *** = P value < 0,001). Right panel shows the quantification of the associated TMEM165 fluorescence intensity (N = 2; n = 50; *** = P value < 0,001).

Figure 3. TMEM165 is targeted to lysosomal degradation after MnCl2 exposure

(A) HeLa cells were treated for 8h with MnCl2 500 μM and/or chloroquine 100 μM, fixed and labeled with antibodies against TMEM165 (upper panels) and LAMP2 (middle panels) before confocal microscopy visualization. (B) Western blot analysis of the same experiment described in (A). Total cell lysates were prepared, subjected to SDS-PAGE and Western blot with the indicated antibodies. Right panel shows the quantification of TMEM165 protein level after normalization with actin (N = 2; *** = P value < 0,001).

Figure 4. TMEM165 lysosomal degradation is Rab7 and Rab5 independent

HeLa cells were transfected with empy vector (mock), Rab7T22N-GFP or Rab5Q79L-GFP. 36H after transfection, cells were treated or not for 4h with MnCl2 500 μM. (A) Total cell lysates were prepared, subjected to SDS-PAGE and Western blot with the indicated antibodies. Right panel shows the quantification of TMEM165 protein levels after normalization with actin (N = 2; *** = P value < 0,001). (B) Immunofluorescence analysis by confocal microscopy of the same experiments as described in (A) (N = 2 ; n= 50).

Figure 5. The TMEM165 (69–172) domain confers Mn sensitivity

(A) TMEM165 full length, short isoform and long isoform protein organization. Grey boxes represent transmembrane domains and the red boxes are the well conserved acidic motifs (B) HeLa cells were transfected with empty vector, SF or LF for 36h then treated with MnCl2 500 μM for 4h. Total cell lysates were prepared, subjected to SDS-PAGE and Western blot with the indicated antibodies. Lower panel shows the percentage of TMEM165 stability after normalization with actin (N = 2; * = P value < 0,05; *** = P value < 0,001).

Figure 6. The ELDGK motif is crucial for Mn2+ sensitivity

(A) Healthy skin fibroblasts (upper left) and patients skin fibroblasts (lower left) carrying E108G mutation were treated with MnCl2 500μM for 0 to 8h. Total cell lysates were prepared, subjected to SDS-PAGE and Western blot with the indicated antibodies. Right panel shows the quantification of TMEM165 protein levels after normalization with actin (N = 2; *** = P value < 0,001). (B) Healthy skin fibroblasts and patients skin fibroblasts carrying E108G mutation were treated with MnCl2 500μM for 0, 4 and 8h. Cells were then fixed and labeled with antibodies against TMEM165 and GM130 before confocal microscopy visualization (N = 2; *** = P value < 0,001). Lower panel shows the quantification of the associated TMEM165 fluorescence intensity (N = 2; n = 30; *** = P value < 0,001).

Figure 7. Mutations in the ELGDK motif are not functional and insensitive to Mn2+

(A) HEK293 cells were transfected with mutac1 -RFP for 36h then treated with MnCl2 500 μM for 4h. Total cell lysates were prepared, subjected to SDS-PAGE and Western blot with the indicated antibodies. (B) HEK293 control cells and HEK293 KO TMEM165 cells were transfected with empty-vector, wild-type or mutac1 plasmid for 36h. Total cell lysates were prepared, subjected to SDS-PAGE and Western blot with the indicated antibodies.

Figure 8. TMEM165 topology

(A) Representation of TMEM165 predicted topology. The two anti-TMEM165 antibodies (Sigma-Aldrich and Thermo Fisher Scientific) depicted here recognize two different parts of the protein. The Sigma one recognize the cytoplasmic loop between the fourth and fifth transmembrane domains. The Thermo one recognize the short luminal loop between the first and the second transmembrane domain. (B) Cells were fixed with paraformaldehyde 4% and treated as described in materials and methods. Selective permeabilization was done by using triton ×100 or digitonin. Cells were labeled with antibodies against TMEM165 and GM130 before confocal microscopy visualization.

Figure 9. Model for TMEM165 degradation in excess of Mn

The left panel present the steady state TMEM165 levels in physiological conditions. The right panel shows TMEM165 lysosomal degradation in excess of Mn.