Fig 1: Recombinant LACTB2 displays endoribonuclease activity. (A) LACTB2 is an endoribonuclease. Recombinant and purified LACTB2 was incubated with 5? end [32P], or 3? end [32P]Cp, radiolabeled 30 nt-long RNA and unlabeled yeast tRNA. The reaction conditions included incubation at 37°C for 15, 30 and 60 min, followed by the addition of formamide dye and analysis by denaturing gel and autoradiography. Lane (L): nucleotide ladder created by alkaline hydrolysis of the substrate RNA. Lane (1): [32P]—AMP marker of 5? [32P]-labeled RNA, created by digestion with RNase One. Lane (-): RNA incubation for 60 min, with no addition of protein. Forms of the same shape presented to the right of the autoradiogram indicate the matching cleavage products of the complementary 5? and 3? RNAs and can be identified at the sequences presented below the figure, in panel C. (B) LACTB2 does not display exoribonucleolytic activity. LACTB2 was incubated with 40 nt body-labeled [32P]-UTP RNA substrate, with the nucleotide sequence shown in panel C. Lane (L): nucleotide ladder described in panel A. Lane (1): [32P]-UMP marker, prepared by the digestion of body-labeled RNA, using Methanocaldoccous jannaschii RNase J3, at 60°C for 1 h (42). Lane (2): [32P]-UDP marker, obtained by digesting body-labeled RNA with Escherichia coli PNPase. Other lanes are labeled as described for panel A. (C) RNA sequence of the substrates. The cleavage sites corresponding to the shape shown in panel A are indicated. The uridines labeled in the body-labeled RNA used in the experiment described in panel B are colored gray.
Fig 2: Recombinant LACTB2 prefers U, but not G or poly(A). (A) LACTB2 was incubated for 15, 30 and 60 min, at 37°C with poly(GU)12, poly(U)20 or poly(A)20, in the presence of yeast tRNA. The RNA substrates were labeled with [32P] at the 5? end. Lane (L): Nucleotide ladder prepared by alkaline hydrolysis of poly(U)20. Lane (-): RNA substrate incubated with no protein for 60 min. Lanes under the triangle—incubation for 15, 30 and 60 min. Lanes labeled ?motifII: the RNAs were incubated for 60 min with the ?motif II mutated protein in which the six amino acids of motif II were deleted (see Figure 7). Following incubation, the RNA was purified and analyzed by denaturing PAGE and autoradiography. (B) Poly(GU)12 was digested in the presence of yeast tRNA with LACTB2, RNase A (cleaves following U) and RNase T1 (cleaves following G). Lane (-) is the same as in panel A. (C) LACTB2 cleavage products have 3?-OH termini and therefore are sensitive to digestion by the exoribonuclease PNPase. 5? [32P] (GU)12 RNA was digested with the enzymes as indicated on the top. When using two enzymes, the RNA was purified by phenol extraction and EtOH precipitation between the incubations. The full length 24 nt RNA contains a 3?-OH and therefore is sensitive to PNPase. The cleavage products of RNase A have 3?-phosphate while those of Nuclease P1 contain 3?-OH. Some leakage of the digestion products of PNPase to the lane of no protein (-) happened when the gel was loaded with the samples. Nucl. P1: Nuclease P1.
Fig 3: LACTB2 is a mitochondrial, soluble and monomeric protein. (A) Mitochondria localization. HeLa cells were disrupted and the nuclear, cytoplasmic and mitochondria-containing fractions were separated and analyzed for the localization of LACTB2, using an immunoblotting assay. The nucleus-located protein, CPSF3L, was used as marker for nuclear proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a cytosolic marker and cytochrome C (Cyt C) as a mitochondrial marker. (B) Immunoblot analysis of mitochondrial extract following Proteinase K accessibility assay. TX-100, Triton X-100. PK, Proteinase K. PNPase is a mitochondrial protein located mainly in the intermembrane space and a small amount in the matrix. HSP60 is a mitochondrial matrix protein. Tom20 is a mitochondrial outer membrane protein. (C) LACTB2 is a soluble protein. Immunoblotting analysis of mitochondrial extract following alkaline sodium carbonate (Na2CO3) extraction of soluble and peripheral membrane proteins. T, total extract. S, supernatant. P, pellet. Subunit 5 of the ATP synthase (ATP5) was used as a marker for intrinsic membrane proteins and Cyt C as a marker of soluble proteins. (D) Immunoblot analysis of the recombinant LACTB2 (Rec.) and the native protein in isolated mitochondria (Mit.) showing the same size on SDS-PAGE. Therefore, the mitochondria targeting signal is not cleaved upon entering the mitochondria and remain an integral part of the mature protein. (E) LACTB2 is present as a monomer. Soluble proteins of bovine mitochondrial matrix were extracted from bovine liver as described in the methods section. This extract was fractionated on a size exclusion column superdex 200 and analyzed for LACTB2, using an immunoblotting assay. The following proteins were used as size markers: Tyroglobulin (669 kDa), polynucleotide phosphorylase (PNPase) (232 kDa), BSA (67 kDa), ß-lactoglobulin (35 kDa) and ribonuclease A (13 kDa).
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