TABLE 2.

Summary of the multi-factorial effects of the various TNZD ZnT2 mutants

	Method/details	G280R	T312M	E355Q
mRNA level (11)	Location at the gene	Exon 6–7	Exon 7	Exon 8
	Nucleotide change	Guanine 838 to adenine	Cytosine 935 to thymine	Guanine 1063 to cytosine
	Impact on splicing	Reduced splicing efficiency by 75% relative to WT	No deleterious effect expceted	No deleterious effect expceted
Location in ZnT2 protein	Predicted location based on the three-dimensional model	Loop between the last transmembrane (TM) helix and a cytoplasmic domain	Loop between β-sheets of a cytoplasmic domain, being in close proximity to TM helices	Solvent-exposed residue in the C terminus
Protein levels and stability	ZnT2 mutant degradation (11)a	80% Enhanced degradation compared with WT	40% Enhanced degradation compared with WT	60% Enhanced degradation compared with WT
	Protein levels upon stable co-transfection with WT-ZnT2 (Fig. 5)b	Low expression compared with the WT	High expression compared with the WT	Slightly reduced expression compared with the WT
	Mutant protein levels under stable transfection (Fig. 8)c	Slightly reduced expression compared with the WT	Slightly reduced expression compared with the WT	Very low expression compared with the WT
	BiFC assay (Fig. 1)	Same YFP intensity levels as the WT	Same YFP intensity levels as the WT	Same YFP intensity levels as the WT
	Calculated thermodynamic destabilization (Table III)	Very unstable	Stable	Unstable
Subcellular localization of ZnT2	Mutant-mutant pairs-BiFC assay (Fig. 2)	Nearly all cells displayed ER localization	Half of the cells showed ER localization and half vesicular localization	Most of the cells showed ER localization
	Mutant-WT pairs-BiFC assay (Fig. 2)	Some vesicular localization was observed in most cells	Most cells preserved vesicular localization	Half of the cells showed ER localization and half vesicular localization
Dimerization	Immunoprecipitation analysis (11)	Forms homodimers with the WT protein	Forms homodimers with the WT protein	Form Homodimers with the WT protein
	BiFC assay (Fig. 1)	Forms homodimers at the same level of the WT protein	Forms homodimers at the same level of the WT protein	Form homodimers at the same level of the WT protein
Function	Zinc toxicity assay	Mutant non-functional; restored the ability to protect cells upon co-expression with WT-ZnT2	Mutant non-functional; restored the ability to protect cells upon co-expression with WT-ZnT2	Mutant non-funct ional; restored the ability to protect cells upon co-expression with WT-ZnT2
	Zinpyr-1 accumulation	Mutant non-functional	Mutant non-functional	Mutant non-functional
	Zinquin accumulation	Mutant non-functional. Yet, did not completely abolish the ability of the WT-ZnT2 protein to accumulate zinc	Mutant non-functional. Yet, did not completely abolish the ability of the WT-ZnT2 protein to accumulate zinc	Mutant non-functional. Yet, did not completely abolish the ability of the WT-ZnT2 protein to accumulate zinc
Zinc coordination	Based on the three-dimensional model	Close to zinc coordination site B in YiiP	Surrounded by conserved polar amino acids that are probably important for zinc transport	Direct coordination of zinc, on the equivalent site C of Y iiP
Zinc levels in breast milk (time after birth)	Normal levels: 80 ± 30 μg/dl at 4–6 months (11)	10 μg/dl (4 months)	<10 μg/dl (6 months)	21 μg/dl (4 months)
Age of infant upon TNZD onset (11)		3 months	5 months	1.5 months

^a Determined by Western blotting analysis after cycloheximide treatment of the different mutants that were stably transfected into DT40 ZnT1^−/− MT^−/−ZnT4^−/− cells.

^b Determined by Western blot analysis of the different mutants that were stably co-transfected with WT-ZnT2 into DT40 ZnT1^−/− MT^−/− ZnT4^−/− cells.

^c Determined by Western blot analysis of the different mutants that were stably transfected into DT40 ZnT1^−/− MT^−/− ZnT4^−/− cells.