Purification of mitochondrial proteins HSP60 and ATP synthase from ascidian eggs: implications for antibody specificity

Abstract

Use of antibodies is a cornerstone of biological studies and it is important to identify the recognized protein with certainty. Generally an antibody is considered specific if it labels a single band of the expected size in the tissue of interest, or has a strong affinity for the antigen produced in a heterologous system. The identity of the antibody target protein is rarely confirmed by purification and sequencing, however in many cases this may be necessary. In this study we sought to characterize the myoplasm, a mitochondria-rich domain present in eggs and segregated into tadpole muscle cells of ascidians (urochordates). The targeted proteins of two antibodies that label the myoplasm were purified using both classic immunoaffinity methods and a novel protein purification scheme based on sequential ion exchange chromatography followed by two-dimensional gel electrophoresis. Surprisingly, mass spectrometry sequencing revealed that in both cases the proteins recognized are unrelated to the original antigens. NN18, a monoclonal antibody which was raised against porcine spinal cord and recognizes the NF-M neurofilament subunit in vertebrates, in fact labels mitochondrial ATP synthase in the ascidian embryo. PMF-C13, an antibody we raised to and purified against PmMRF, which is the MyoD homolog of the ascidian Phallusia mammillata, in fact recognizes mitochondrial HSP60. High resolution immunolabeling on whole embryos and isolated cortices demonstrates localization to the inner mitochondrial membrane for both ATP synthase and HSP60. We discuss the general implications of our results for antibody specificity and the verification methods which can be used to determine unequivocally an antibody's target.

Figure 1. Segregation of the mitochondria-rich myoplasm into the muscle lineage of the ascidian embryo.

Green speckles indicate mitochondria; the violet oval depicts the cortical domain rich in RNA, endoplasmic reticulum and germ plasm known as the CAB (for centrosome attracting body). (A) Drawings of relevant stages of the bilaterally symmetric ascidian embryo. Descendents of the B4.1 vegetal posterior blastomere are labeled. The 2 and 8 cell stages show posterior views; 16, 32, 64 cell stages are vegetal views. (B) a lateral view of the 8 cell stage and lineage of the primary muscle cells. At the 64 cell stage the bulk of the myoplasm partitions into 3 pairs of cells (B7.4, B7.5, B7.8) which give rise to the primary muscle of the tadpole tail, and the CAB domain segregates into B7.6germ line cells. Concerning myoplasm distribution during cell division, there are 3 “equal cleavages” whereby myoplasm is inherited by both daughter cells (1st cleavage, B4.1, and B5.2) and there are 6 “unequal cleavages” whereby myoplasm distributes preferentially to one daughter cell (B5.1, B6.2, B6.3, B6.4 as well as 2nd and 3rd cleavages which are not shown).

Figure 2. Distribution of p58 during ascidian embryonic development.

(A) Immunoblot using NN18 antibody on Phallusia mammillata extracts prepared at the indicated stages of development; hr: hours after fertilization. (B) Examples of Phallusia embryos stained with NN18; egg: an equatorial view (top) or surface view (bottom) of the same unfertilized egg; 16 cell: 2 different embryos with 4 posterior cells (B5.1 and B5.2 pairs) labeled. Gastrula: posterior muscle territory is labeled. Tailbud and tadpole tail: individual mononucleate muscle cells are distinguishable. Scale bars are 20 microns in all panels.

Figure 3. Production of antibody PMF-C13 and distribution of p63 during ascidian embryonic development.

(A) Schematics of PmMRF protein (top line) and four versions produced in bacteria (black bars). bHLH: helix-loop-helix domain diagnostic of MyoD family. The number of amino acids of PmMRF present in each construct is indicated on the right. “GST” indicates that these 2 proteins also contain the 220 amino acids of Glutathione-S-Transferase. The antigenic peptide corresponding to amino acids 496–508 (colored orange) is present in “PMF547” which encodes most of PmMRF, but is absent from the others. (B) Gels containing PmMRF fusion proteins depicted in A were stained with Coomassie blue (left) or immunoblotted with antibody PMF-C13 (right). In each case 1 µg was loaded except for the PMF547 lane of the immunoblot which was loaded with much less protein (10 ng, indicated on the bottom). (C,D) Immunoblots using PMF-C13 antibody on ascidian protein extracts prepared at the indicated stages of development; hr: hours after fertilization. All samples are from Phallusia (Pm) except for three lanes in D which show PMF-C13 also recognizes p63 in Ciona (Ci) extracts. (E) Examples of Phallusia embryos stained with PMF-C13. Scale bars are 20 microns in all panels. The names in white indicate the embryonic stage and the numbers in black indicate the muscle lineage cells at the 4, 8, 16, 32 cell stages (see Fig. 1); the 64 cell stage cell names are indicated on the slightly later 110 cell embryo although B7.4 and B7.8 have already divided. Arrows indicate the cortical CAB which excludes myoplasm and thus is detectable as an unstained region surrounded by myoplasm label.

Figure 4. Purification of p58 by immunoprecipitation.

(A) Beads to which the antibody NN18 was bound were incubated with (+) or without (−)Ciona oocyte homogenate and eluted proteins were subjected to immunoblot (left) or silver stain (right). The major proteins in the immunoprecipitate are p58 (arrow head) and the monoclonal antibody (arrows) which migrates as a large IgG because of the nondenaturing conditions of the gel (see text and methods). (B) N-terminal sequence of immunoprecipitated p58 (bottom line, circled) compared to ATP synthase alpha-subunit from diverse species; asterisks * indicate residues which are identical between p58 protein and Ciona ATP synthase alpha subunit. Immunoprecipitated p58 lacks the first 43 amino acids of ATP synthase which correspond to reported mitochondrial signal peptides (boxed). The “ImmunoScreen” line is a translation of the cDNA clones (NP_001027729) obtained by screening an expression library with NN18 antibody, which is identical to the ATP synthase gene model (KH.C10.579) annotated in the Ciona genome database, except in one position due to a polymorphism. See Fig. S2 for the complete alignment and database accession numbers.

Figure 5. Purification of p63 using successive DEAE ion exchange columns and IEF-PAGE.

In A, C and D, pairs of identical gels show distribution of p63 (western blot with PMF-C13, lower gels) compared to total protein (upper gels). (A) Total Phallusia egg extract prepared at low ionic strength was passed through an ion exchange column, eluted with a step salt gradient and representative fractions were analyzed; p63 (arrowhead) elutes at 100–200 mMNaCl but is not well separated from the majority of proteins. (B) Schematic of biochemical approach used to enrich p63 before 2D-electrophoresis. (C) Scale-up and sequential ion exchange columns. Column 1: The majority of proteins are retained on the overloaded column (“elute peak”) but p63 (arrowhead) passes through. The flow through (FT) from the first column (left) was loaded onto a second column as indicated by the curved arrow. Column 2: p63 elutes at 200 mM salt as in A. A fraction enriched for p63 (black rectangle) was loaded onto the duplicate 2D gels shown in D: the p63 spot (arrow) is abundant and well separated from other proteins.

Figure 6. p58/ATP Synthase and p63/HSP60 localize to all mitochondria in the ascidian egg.

Confocal views of Phallusia eggs labeled in vivo for mitochondria with DiOC₂(3) (A) or fixed and double immunolabelled (B–H) with NN18 (red) or PMF-C13 (green). Eggs are oriented with the animal pole up and the vegetal hemisphere containing the myoplasm basket down. A and B show low magnification views of the center (top) or the surface (bottom) of the same egg. Boxes in B indicate positions zoomed to high magnification to detail the animal pole (C, F), the equatorial region (D,G) or the vegetal myoplasm (E,H). Yellow overlay (right panels) shows colocalization; both antibodies label individual mitochondria (arrows) and PMF-C13 also labels smaller cytoplasmic particles (arrowheads). The animal pole region (ap in C) which contains the meiotic spindle is devoid of mitochondria (as shown by live DiO labeling, inset in C). Scale bars are 10 microns in all panels.

Figure 7. Cortices isolated from fertilized eggs and labeled with DiOC₆(3) to show mitochondria (blue) and with antibodies NN18 (red) or PMF-C13 (green).

(A,B,D) “+ triton”: cortices immunolabeled after permeabilization with detergent. (C,E) “− triton”: cortices immunolabeled in the absence of permeabilization: no antibody signal is detected. Scale bars are 5 microns.