Overton JD , Komiya Y , Mezzacappa C , Nama K , Cai N , Lou L , Fedeles SV , Habas R , Runnels LW .

R01 GM080753 NIGMS NIH HHS , GM078172 NIGMS NIH HHS , GM080753 NIGMS NIH HHS , R01 GM078172 NIGMS NIH HHS , S10 OD010572 NIH HHS

	Figure 1: Hepatocystin interacts with TRPM7. (A) Schematics of the linear structures and domains of TRPM7 and hepatocystin. TRPM7 contains an NH2-terminus melastatin domain, six transmembrane domain, TRP box, as well as coiled-coil (CC), serine/threonine rich domain (ST), and functional alpha kinase (KIN) domains. Hepatocystin contains a leader signal peptide (SP), a low-density lipoprotein class A (LDL) domain, two Ca2+-binding EF hands, a glutamic acid-rich segment (GLUT), a region with low homology to the mannose 6-phosphate receptor (MRH), and a COOH-terminal His-Asp-Glu-Leu (HDEL) ER retention signal. (B) Directed yeast-two-hybrid assay using the indicated fragments of TRPM7 COOH-terminus fused to LexA as bait and Gal4 fused residues 346-528 of hepatocystin as prey. LexA fused to Laminin (Lex-Lam) served as a negative control. (C) Pulldown purification assay using GST fused to residues 346-528 of hepatocystin (GST-HepCT) against HEK-293 cell lysates containing the indicated mfGFP-tagged TRPM7 COOH-terminal fragments. (D) Endogenous hepatocystin co-immunoprecipitates with HA-tagged TRPM7 heterologously expressed in HEK-293T cells. (E) Immunocytochemistry of HEK-293T cells transfected with HA-tagged TRPM7 and FLAG-tagged hepatocystin (Green) show that the two proteins co-localize in the endoplasmic reticulum (ER). A scale bar of 20 microns in length is indicated.
	Figure 3: Xhepatocystin is required for gastrulation. (A) Schematic diagram of Xhepatocystin morpholino (MO) binding sites upstream and overlapping the start site of Xhepatocystin. (B) Western blot analysis shows that injection of the Xhepatocystin MO (50 and 75 ng), but not the control MO (75 ng), can effectively inhibit translation of Myc-Xhepatocystin 5′ UTR injected RNA (250 pg). β-tubulin is shown as a loading control. (C) Injection of Xhepatocystin MO into the dorsal blastomeres of the 4-cell stage embryos inhibited gastrulation. The gastrulation defect phenotype was rescued by co-injection of mouse hepatocystin RNA and mouse TRPM7 RNA (125 pg), but not LacZ RNA (125 pg), with the Xhepatocystin MO (75 ng). Injection of the control MO (75 ng) produced no phenotype. (D) Quantification of phenotypic results from (C). Phenotypes were scored according to the severity of the gastrulation defect (GD) at the tadpole stage (E). The injections were repeated at least three times. The ability of co-injection of mouse hepatocystin and mouse TRPM7 RNA with Xhepatocystin MO to rescue the gastrulation phenotype caused by the Xhepatocystin MO was statistically significant (*P<0.05). The collective total number of injected embryos from all experiments is indicated above each bar.
	Figure 4: TRPM7 and hepatocystin functionally interact. (A,B) Co-injection of Xhepatocystin MO (15 ng) and XTRPM7 MO (15 ng) synergistically inhibit gastrulation, but have little or no effect when injected separately. (B) Phenotypes were scored according to the severity of the gastrulation defect (GD) at the tadpole stage. The dotted line in (B) indicates the amount of gastrulation defects that would be expected if the effects were additive (43%). The injections were repeated three times. Statistic analysis indicated that the number of embryos exhibiting gastrulation defects (77%) by co-injection of Xhepatocystin and XTRPM7 MOs was significantly different (P=0.05) from what would be expected if the effects were additive (43%). The collective total number of injected embryos from all three experiments is indicated above each bar.
	Figure 5: Hepatocystin and N-glycosylation affect the abundance of TRPM7. (A) Treatment of cells with tunicamycin (5 μg/ml) for 24 hours decreased tetracycline-induced TRPM7 protein expression in 293-TRPM7 cells. (B) Quantification of data from (A). (C) Co-injection of increasing dosages of the Xhepatocystin MO with 250 pg mRNA of HA-tagged mTRPM7 (HA-TRPM7) decreases TRPM7 expression in Xenopus embryos at stage 17. β-actin is shown as a loading control. (D) Quantification of data from (C). The experiments were repeated at least three times.
	prkcsh (protein kinase C substrate 80K-H) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	prkcsh (protein kinase C substrate 80K-H) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	Figure 1. Hepatocystin interacts with TRPM7.(A) Schematics of the linear structures and domains of TRPM7 and hepatocystin. TRPM7 contains an NH2-terminus melastatin domain, six transmembrane domain, TRP box, as well as coiled-coil (CC), serine/threonine rich domain (ST), and functional alpha kinase (KIN) domains. Hepatocystin contains a leader signal peptide (SP), a low-density lipoprotein class A (LDL) domain, two Ca2+-binding EF hands, a glutamic acid-rich segment (GLUT), a region with low homology to the mannose 6-phosphate receptor (MRH), and a COOH-terminal His-Asp-Glu-Leu (HDEL) ER retention signal. (B) Directed yeast-two-hybrid assay using the indicated fragments of TRPM7 COOH-terminus fused to LexA as bait and Gal4 fused residues 346-528 of hepatocystin as prey. LexA fused to Laminin (Lex-Lam) served as a negative control. (C) Pulldown purification assay using GST fused to residues 346-528 of hepatocystin (GST-HepCT) against HEK-293 cell lysates containing the indicated mfGFP-tagged TRPM7 COOH-terminal fragments. (D) Endogenous hepatocystin co-immunoprecipitates with HA-tagged TRPM7 heterologously expressed in HEK-293T cells. (E) Immunocytochemistry of HEK-293T cells transfected with HA-tagged TRPM7 and FLAG-tagged hepatocystin (Green) show that the two proteins co-localize in the endoplasmic reticulum (ER). A scale bar of 20 microns in length is indicated.
	Figure 3. Xhepatocystin is required for gastrulation.(A) Schematic diagram of Xhepatocystin morpholino (MO) binding sites upstream and overlapping the start site of Xhepatocystin. (B) Western blot analysis shows that injection of the Xhepatocystin MO (50 and 75 ng), but not the control MO (75 ng), can effectively inhibit translation of Myc-Xhepatocystin 5′ UTR injected RNA (250 pg). β-tubulin is shown as a loading control. (C) Injection of Xhepatocystin MO into the dorsal blastomeres of the 4-cell stage embryos inhibited gastrulation. The gastrulation defect phenotype was rescued by co-injection of mouse hepatocystin RNA and mouse TRPM7 RNA (125 pg), but not LacZ RNA (125 pg), with the Xhepatocystin MO (75 ng). Injection of the control MO (75 ng) produced no phenotype. (D) Quantification of phenotypic results from (C). Phenotypes were scored according to the severity of the gastrulation defect (GD) at the tadpole stage (E). The injections were repeated at least three times. The ability of co-injection of mouse hepatocystin and mouse TRPM7 RNA with Xhepatocystin MO to rescue the gastrulation phenotype caused by the Xhepatocystin MO was statistically significant (*P<0.05). The collective total number of injected embryos from all experiments is indicated above each bar.
	Figure 4. TRPM7 and hepatocystin functionally interact.(A,B) Co-injection of Xhepatocystin MO (15 ng) and XTRPM7 MO (15 ng) synergistically inhibit gastrulation, but have little or no effect when injected separately. (B) Phenotypes were scored according to the severity of the gastrulation defect (GD) at the tadpole stage. The dotted line in (B) indicates the amount of gastrulation defects that would be expected if the effects were additive (43%). The injections were repeated three times. Statistic analysis indicated that the number of embryos exhibiting gastrulation defects (77%) by co-injection of Xhepatocystin and XTRPM7 MOs was significantly different (P=0.05) from what would be expected if the effects were additive (43%). The collective total number of injected embryos from all three experiments is indicated above each bar.
	Figure 5. Hepatocystin and N-glycosylation affect the abundance of TRPM7.(A) Treatment of cells with tunicamycin (5 μg/ml) for 24 hours decreased tetracycline-induced TRPM7 protein expression in 293-TRPM7 cells. (B) Quantification of data from (A). (C) Co-injection of increasing dosages of the Xhepatocystin MO with 250 pg mRNA of HA-tagged mTRPM7 (HA-TRPM7) decreases TRPM7 expression in Xenopus embryos at stage 17. β-actin is shown as a loading control. (D) Quantification of data from (C). The experiments were repeated at least three times.

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