Defining the protein complex proteome of plant mitochondria

Abstract

A classical approach, protein separation by two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was combined with tandem mass spectrometry and up-to-date computer technology to characterize the mitochondrial "protein complex proteome" of Arabidopsis (Arabidopsis thaliana) in so far unrivaled depth. We further developed the novel GelMap software package to annotate and evaluate two-dimensional blue native/sodium dodecyl sulfate gels. The software allows (1) annotation of proteins according to functional and structural correlations (e.g. subunits of a distinct protein complex), (2) assignment of comprehensive protein identification lists to individual gel spots, and thereby (3) selective display of protein complexes of low abundance. In total, 471 distinct proteins were identified by mass spectrometry, several of which form part of at least 35 different mitochondrial protein complexes. To our knowledge, numerous protein complexes were described for the first time (e.g. complexes including pentatricopeptide repeat proteins involved in nucleic acid metabolism). Discovery of further protein complexes within our data set is open to everybody via the public GelMap portal at www.gelmap.de/arabidopsis_mito.

Figure 1.

Analysis of the mitochondrial proteome of Arabidopsis by 2D BN/SDS-PAGE. Freshly isolated mitochondria (1 mg of mitochondrial protein) were solubilized by digitonin (5 g detergent g⁻¹ protein), supplemented with Coomassie blue and loaded onto the 2D gel system. After electrophoresis, the gel was stained by Coomassie blue-colloidal. The identity of the OXPHOS complexes is given above the gel. I+III₂, Supercomplex composed of complex I and dimeric complex III; I, complex I; V, complex V (ATP synthase); III₂, dimeric complex III; F₁, F₁ part of ATP synthase; F₀, F₀ part of ATP synthase; IV, complex IV; II, complex II.

Figure 2.

Molecular mass scale for the gel shown in Figure 1. The numbers refer to molecular masses (in kD). Masses used for calibration are given in red. BN gel dimension (horizontal) is as follows: I+III₂ supercomplex (1,500 kD), complex I (1,000 kD), dimeric complex III (500 kD), complex II (160 kD), and aconitate hydratase (110 kD). SDS gel dimension (vertical) is as follows: 75-kD subunit of complex I (spot 171), α-MPP (52 kD; spot 47), prohibitin (30 kD; spot 175), FeS subunit of complex III (23 kD; spot 53), QCR7 subunit of complex III (14 kD; spot 54), and the QCR8 subunit of complex III (8 kD; spot 56). Masses between those used for calibration were calculated with Eureqa software (version 0.83 β; www.eureqa.com).

Figure 3.

Reference map of the protein complex proteome of Arabidopsis mitochondria as presented in GelMap (www.gelmap.de/arabidopsis_mito). Protein spots identified by MS are circled. Most spots include multiple proteins. The menu to the right offers functional classifications of subunits according to physiological functions and protein complexes. The numbers in parentheses refer to the number of identified proteins per individual complex. Upon clicking on a protein complex, names of all its subunits become available (Supplemental Fig. S2). Detailed information on spots is given upon clicking on the encircled spots on the 2D image (see Fig. 4). The GelMap software package also has different search options (e.g. for a distinct protein by name or accession number). The search options are located beneath the menu to the right of the gel image (not shown here). For further features of GelMap, see www.gelmap.de/arabidopsis_mito.

Figure 4.

Pop-up function on the gel map. Information about the proteins identified in a spot can be viewed directly on the gel image. The user can hover over a spot (1) that opens a tooltip containing the names of all included proteins (left) as well as their MS reliability scores (right). By clicking on the spot (2), the window is fixed, and protein names are hyperlinked to more detailed information. Protein details become visible in a new window by clicking on the protein of interest (3). If only one protein is identified in a spot, the first click on the spot already opens the details window shown in 3. This window contains (from top to bottom): spot number (in the blue frame), protein name, dedicated protein complex, MS reliability score, the calculated molecular mass, and the Accession number (according to TAIR), which is linked to the respective entry on the TAIR homepage (www.arabidopsis.org). The “more protein details” link at the bottom of the details window is linked to a table showing all proteins identified in the respective spot (Supplemental Table S1). Besides the characteristics already given in the pop-up window, it contains further information about spot characteristics (such as coordinates and apparent masses), MS analysis, and the physiological context of the proteins (Table I). The “more peptide details” link leads to a table that includes details of the peptides identified by MS.