PMC

Characterization of the Pof1 protein. A, cellular localization of Pof1 in yeast cells. The fluorescence signal of Pof1-GFP along with the differential interference contrast (DIC) and DAPI (which marks the nuclear and mitochondrial DNA) images indicate that Pof1 localizes both in the cytoplasm and the nucleus. Direct genomic integration of a GFP tag (upstream of the POF1 ORF) into haploid WT yeast genome does not interfere with the normal function of Pof1, because unlike the pof1Δ mutant, the GFP-tagged strain releases WT level of NR, determined by cross-feeding plate assay (bottom panel). B, rPof1 protein catalyzes the conversion of NMN to NAD⁺. The Michaelis-Menten plot of rPof1 is shown. The apparent kinetic parameters of rPof1 protein are shown in the table. C, Pof1 exhibits NMNAT activity in vivo. The results show that POF1 is required for the growth of nma1Δnma2Δ mutants on NR-supplemented medium. Cells lacking both NMA1 and NMA2 require NR for viability (panels I and II). 5-FOA is toxic to cells carrying the URA3 gene (panel III), which therefore forces cells to lose the URA3 plasmid for viability. Loss of the POF1-expressing plasmid (POF1-URA3) in the triple deletion mutant nma1Δnma2Δpof1Δ results in lethality even in the presence of NR (panel III, bottom panel), suggesting that Pof1 is indispensable in the absence of other NMNATs. D, Nrk1 overexpression rescues the high NR release phenotype of the pof1Δ mutant. E, Pof1 does not have NR kinase function. The results show that NRK1 is required for the growth of nma1Δnma2Δ on NR-supplemented medium. The loss of the NRK1-expressing plasmid (NRK1-URA3) in the triple deletion mutant nma1Δnma2Δnrk1Δ results in lethality even in the presence of NR (and POF1) (panel III, bottom panel), suggesting that Nrk1 is the only NR kinase in budding yeast. Exp, experiment.

Immunocytochemistry assays to study Pof1 protein cell localization and distribution. The POF1 null cells were used as a negative control to establish antibody background levels.

Secretion of progesterone from POFs. (A-B) qRT-PCR analysis of Cyp11a1 and Hsd3b2 mRNA abundance in POFs (POF1 to POF5). (C-D) WB analysis of CYP11A1 expression in the POFs (POF1 to POF5). (E) The level of P₄ showed in F1 and POFs (POF1 to POF5). Values are means ± SEM of three experiments.

Caspase-induced apoptosis leads to POF degradation. (A-B) WB and grey analysis of caspase3 expression in POFs (POF1 to POF4). Values are means ± SEM of three experiments. Asterisks indicate significant differences (* P<0.05 and ** P<0.01). (B-E) Few TUNEL (green) marker labeled occurred in cells from POF1 to POF3. (D) Negative Control. Scale bar: 50 µm.