GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain
- Albert Einstein College of Medicine, United States
- University of Oulu, Finland
- University Medical Center Hamburg-Eppendorf, Germany
Figures
Figure 1
Expression constructs.
Mouse GnT1IP-L (417 aa) contains an N-terminal cytoplasmic domain of 48 aa, a transmembrane (TM) domain of 21 aa (shaded), and a luminal domain of 348 amino acids. The location of the Myc tag (red) …
Figure 2
The luminal domain of GnT1IP-L inhibits MGAT1.
(A) HeLa cells transiently expressing the chimera MGAT1/GnT1IP-L-Myc or MGAT1/GnT1IP-L-Myc-KDEL were analysed for expression of Myc, MAN2A1, GM130 and protein disulfide isomerase (PDI). Each result …
Figure 3
The TM and cytoplasmic domain of GnT1IP-L does not inhibit MGAT1.
(A) Lectin-resistance test of cloned CHO cells stably expressing GnT1IP-L/MGAT1-Myc compared to CHO cells and Lec1 CHO cells that lack MGAT1 (n = 2). (B) The same cloned GnT1IP-L transfectant lines …
Figure 4
GnT1IP-L interacts specifically with MGAT1 in the Golgi.
(A) Fluorescence microscopy of COS-7 cells stably expressing GnT1IP-L-mVen and transiently expressing medial Golgi GlcNAc-transferases MGAT1 to MGAT5 conjugated to mChe at their C-terminus compared …
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Figure 4—source data 1
GnT1IP-L interactions with human and mouse MGATs in the Golgi of COS-7 and CHO Lec1 cells.
- https://doi.org/10.7554/eLife.08916.010
Figure 5
ER-localized GnT1IP-L does not inhibit MGAT1.
(A) HeLa cells transiently expressing GnT1IP-L-Myc and MGAT1-HA, or the chimera Iv/GnT1IP-L-Myc with and without MGAT1-HA were analysed for expression of Myc, HA and PDI. Each result is …
Figure 6
GnT1IP-L and MGAT1 form homomers in the endoplasmic reticulum (ER) and heteromers in the Golgi.
(A) COS-7 cells were co-transfected with mouse GnT1IP-L-VN and GnT1IP-L-VC and, after 8 hr in culture, one set of plates was treated with 1 µg/ml nocodazole overnight. Cells were examined by …
Figure 7
Golgi-localized GnT1IP-L and MGAT1 heteromers are disrupted in the ER following nocodazole treatment and reform after recovery.
(A) COS-7 cells stably expressing GnT1IP-L-mVen were transfected with human MGAT1-mChe and, after 16 hr, treated with 1 µg/ml nocodazole. After 4 hr of treatment, nocodazole was removed from half …
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Figure 7—source data 1
Disruption of Golgi-localized GnT1IP-L and MGAT1 heteromers in the ER following nocodazole treatment and their recovery in the Golgi after drug removal.
- https://doi.org/10.7554/eLife.08916.013
Figure 8
Chloroquine inhibits the formation of GnT1IP-L and MGAT1 heteromers and favors homomers.
(A) COS-7 cells stably expressing GnT1IP-L-mVen were transfected with mouse GnT1IP-L-mChe or human MGAT1-mChe and treated after 8 or 16 hr with 40 μM chloroquine for 16 hr or 4 hr, respectively. …
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Figure 8—source data 1
Disruption of GnT1IP-L and MGAT1 heteromers and enhanced formation of homomers following chloroquine treatment.
- https://doi.org/10.7554/eLife.08916.015
Figure 9
RNA-Seq data for GnT1IP/Mgat4d and Mgat1 in mouse germ cells.
Histogram overlay plot for GnT1IP/Mgat4D (blue) and Mgat1 (red) gene expression in isolated mouse germ cell subtypes as described in Soumillon et al. (2013a). (A) Sertoli cells, (B) Spermatogonia, (C…
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Figure 9—source data 1
R code and comma-delimited data files for generating Figure 9.
The R code files import the data files and exactly reproduce Figure 9. The data file contains the log2 FPKM data for the different testicular cell types from Soumillon et al. (Cell Reports 3, 2179–2190, 2013).
- https://doi.org/10.7554/eLife.08916.017
Figure 10
GnT1IP/MGAT4D and MGAT1 transcripts in testis biopsies from men with impaired spermatogenesis.
Transcript levels of GnT1IP/MGAT4D and MGAT1 were determined from the microarray data of Spiess et al. (2007). (A) Boxplot of GnT1IP/MGAT4D log2 fluorescence in human testicular biopsies presenting …
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Figure 10—source data 1
R code and comma-delimited data files for generating Figure 10.
The R code files import the data files and exactly reproduce Figure 10. The data file contains the log2-transformed expression values for MGAT4D and MGAT1 for sample replicates of different spermatogenic arrest states, as described in Spiess et al. (Hum Reprod 22, 2936–2946, 2007).
- https://doi.org/10.7554/eLife.08916.019
Tables
Table 1
Primers for expression constructs
| GnT1IP-L-Myc |
| For: 1301: (HindIII, Kozak) CAGATCAAGCTTCCACCATGTGCCTGGGAGAAAGTGTTGGGGACC |
| Rev: 1346: (Myc, BamH1) GACTAGGGATCCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGTAATAATTATCCTTGAGGTGC |
| Myc-GnT1IP-L |
| For: 1312: (HindIII, Kozak, Myc) CAGATCAAGCTTCCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGTGCCTGGGAGAAAGTGTTGGGGACC |
| Rev: 1313: (BamH1) GCCTGTGGATCCCTAGTAATAATTATCCTTGAGGTGCTG |
| HA-GnT1IP-L |
| For: 1068: (HindIII, Kozak, HA-GnT1IP-L) GGAACTAAGCTTCCACCATGTACCCTTATGACGTCCCCGATTACGCCAGCCTGTGCCTGGGAGAAAGTG TTGGGG |
| Rev: 1313 |
| GnT1IP-L (F/L) |
| For: 1431: CTCTTAGCCTTAGTTGCCGTCCTGCTCTTAGGTTTATCGTGTTTATGC |
| Rev: 1432: GCATAAACACGATAAACCTAAGAGCAGGACGGCAACTAAGGCTAAGAG |
| GnT1IP-L (F/A) |
| For: 1467: CTCGCCGCCGCCGTTGCCGTCCTGCTCGCTGGTGCCTCGTGTGCCTGC |
| Rev: 1468: GCAGGCACACGAGGCACCAGCGAGCAGGACGGCAACGGCGGCGGCGAG |
| GnT1IP-L-Myc-KDEL |
| For: 1301 |
| Rev: 1471: (BamH1 Myc-KDEL) GGATCCCTACAACTCATCTTTCAGATCCTCTTCTGAGATGAG |
| MGAT1/GnT1IP-L-Myc |
| For: 1282 (HindIII, Kozak) GGACCGAAGCTTCCACCATGCTGAAGAAGCAGTCTGCAGGGC |
| Internal: MGAT1/GnT1IP-L |
| Rev: 1434: TTGATTATTGGTTTGGTTCATCCTCCAGAAGAAGAGGAGCAGCAG |
| For: 1433: CTGCTGCTCCTCTTCTTCTGGAGGATGAACCAAACCAATAATCAA |
| Rev: 1346: (BamH1, Myc) GACTAGGGATCCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGTAATAATTATCCTTGAGGTGC |
| MGAT1/GnT1IP-L-Myc-KDEL |
| For: 1282 |
| Rev: 1471: (BamH1, Myc-KDEL) GGAGTCGGATCCCTACAACTCATCTTTCAGATCCTCTTCTGAGATGAG |
| Iv/GnT1IP-L-Myc |
| For: 1435: (HindIII, Kozak) GGACCGAAGCTTCCACCATGCACAGGAGGAGAAGCAGG |
| Internal Iv/GnT1IP-L |
| Rev: 1436: CAAGTTAACGTTCTTGGCCTTCATTCCGCGGCTGCACTTGCTCTC |
| For: 1437: GAGAGCAAGTGCAGCCGCGGAATGAAGGCCAAGAACGTTAACTTG |
| Rev: 1346: (BamH1, Myc) |
| Iv/GnT1IP-L-Myc-KDEL |
| For: 1435 |
| Rev: 1471 |
| GnT1IP-L/MGAT1-Myc |
| For: 161: (Kozak) GCCACCATGTGCCTGGGAGAAAGTGTTGGGGACCTG |
| Internal (MGAT1/GnT1IP-L) |
| Rev: 162: GTCTGAGGGCAGCCTGCCAGGTGCTGGGCGGGAGATGCAGAAACACGAGAAACCAAAGAG |
| For: 163: CTCTTTGGTTTCTCGTGTTTCTGCATCTCCCGCCCAGCACCTGGCAGGCTGCCCTCAGAC |
| Rev: 164: (MGAT1-Myc) CTACAGATCTTCTTCAGAAATAAGTTTTTGTTCATTCCAGCTAGGATCATAGCCAGTCCATGT |
Table 2
Glycosyltransferase activities in CHO cells expressing HA-GnT1IP-L
| Cells | B4GALT1 nmol/mg/hr | MGAT1 nmol/mg/hr | MGAT2 nmol/mg/hr | MGAT5 nmol/mg/hr |
|---|---|---|---|---|
| CHO Ratio to B4GALT1 | 15.6 (11–23.8) – | 4.2 (3.2–5.5) 0.27 | 0.41 (0.4–0.42) 0.026 | 0.34 (0.35–0.48) 0.022 |
| CHO/HA-GnT1IP-L Ratio to B4GALT1 | 11.5 (9.7–13.2) – | 1.1 (0.9–1.3) 0.096 | 0.38 (0.16–0.61) 0.033 | 0.33 (0.24–0.41) 0.028 |
| Ratio activity GnT1IP-L:CHO | 0.74 | 0.26 | 0.93 | 0.97 |
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Glycosyltransferase assays were performed as described in ‘Materials and methods’ on cell extracts from CHO cells and CHO cells stably expressing HA-GnT1IP-L. Each assay was performed in duplicate, and activity (with range) is given as the average of duplicates for 2–3 independent assays.
Additional files
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Source code 1
Glycosyltransferase assay data for CHO cells expressing HA-GnT1IP-L.
- https://doi.org/10.7554/eLife.08916.020
