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. 2015 Apr 27;112(19):6050–6055. doi: 10.1073/pnas.1424017112

Fig. 5.

Fig. 5.

SLC25A33 gene encoding for mitochondrial pyrimidine nucleotide carrier is majorly responsible for UDP-GlcNAc incorporation to mitochondrial matrix. (A) Transport assay on phosphatidylcholine (PC)-based preformed proteoliposome made without PC extrusion (blue), with PC extrusion and protein (red), and with 1% cholesterol + PC with extrusion and protein (green). DNC samples contain deoxyribonucleotide carrier and PNC samples contain pyrimidine nucleotide carrier. +I samples contain 20 mM bathophenanthroline. NBC samples contain the cell membrane sodium bicarbonate transporter as a negative control. (B) Transport assay with internal substrate to assess antiporter activity of pnc protein. Control sample contains no protein and others contain ATP, ADP, UDP, and UTP inside preformed proteoliposome. (C) Knockdown of SLC25A33 protein from HeLa cells using 500 pmol (0.5) and 2 nmol (2) of stealth siRNA. Control samples contain 500 pmol control siRNA. Both whole cell lysate and mitochondrial fraction are run on gel to analyze total O-GlcNAc, pnc1, and mito complex I levels. (D) Quantification of mito-enriched fractions of O-GlcNAc blot from C above against complex I (n = 3). (E) Transport assay of radioactive UDP-GlcNAc uptake in enriched mitochondria prepared from HeLa cells after treatment with control and pnc siRNA (n = 3). (*P < 0.05).