Co-localization of glyceraldehyde-3-phosphate dehydrogenase with ferredoxin-NADP reductase in pea leaf chloroplasts

Abstract

In immunogold double-labeling of pea leaf thin sections with antibodies raised against ferredoxin-NADP reductase (EC 1.18.1.2, FNR) and antibodies directed against the A or B subunits of the NADP-linked glyceraldehyde-3-P dehydrogenase (GAPD) (EC 1.2.1.13), many small and large gold particles were found together over the chloroplasts. Nearest neighbor analysis of the distribution of the gold particles indicates that FNR and the NADP-linked GAPD are co-localized, in situ. This suggests that FNR might carry FADH2 or NADPH from the thylakoid membrane to GAPD, or that ferredoxin might carry electrons to FNR co-localized with GAPD in the stroma. Crystal structures of the spinach enzymes are available. When they are docked computationally, the proteins appear, as modeled, to be able to form at least two different complexes. One involves a single GAPD monomer and an FNR monomer (or dimer). The amino acid residues located at the putative interface are highly conserved on the chloroplastic forms of both enzymes. The other potential complex involves the GAPD A2B2 tetramer and an FNR monomer (or dimer). The interface residues are conserved in this model as well. Ferredoxin is able to interact with FNR in either complex.

Fig. 1

Micrograph showing a portion of a chloroplast in a pea leaf section doubly labeled with antibodies directed against GAPD (20 nm particles) and with antibodies raised against FNR (10 nm particles). Bar = 200 nm. S, stroma; T, thylakoid; Cy, cytosol; Cw, cell wall. The maximum possible distance between the centers of two gold particles marking GAPD and FNR molecules that are in direct contact with one another would be about 86 nm (diameter of the two protein molecules, four IgG molecules and the radii of the two gold particles). Note high incidence of particles marking FNR over regions of the stroma distant from the thylakoid membranes.

Fig. 2

(a) Plot of the negative log of 1 - fraction in the ordered list of measurements against the square of the distance between nearest neighbor gold particles marking GAPD subunit A and gold particles marking FNR, from the experiment in Fig. 1. There were 555 measurements. The first 271 data points are shown. 104 of the 555 gold particles marking GAPD A are non-randomly distributed with respect to gold particles marking FNR. 392 of the 555 particles marking GAPD A would be close enough to a particle marking FNR to represent adjacent proteins, if one assumes that the four antibodies and two antigens are arranged in linear fashion (i.e. maximum distance separating the two gold particles marking adjacent antigens). (b) Plot of the negative log of 1 - fraction in the ordered list of measurements against the square of the distance between nearest neighbor gold particles when the particle sizes were reversed. There were 284 measurements. The first 162 data points are shown. Fifty five of the 284 gold particles marking FNR are non-randomly distributed with respect to gold particles marking GAPD A, and 129 of the 284 particles would be close enough to the gold particles marking GAPD to indicate adjacent antigens (on the basis of the maximum possible distance separating the gold particles). In an earlier experiment (Anderson et al., 2003) antigens recognized by preimmune rabbit and sheep antisera were distributed randomly with respect to one another.

Fig. 3

(a) Plot of the negative log of 1 - fraction in the ordered list of measurements against the square of the distance between nearest neighbor gold particles marking GAPD subunit B and gold particles marking FNR. There were 286 measurements. The first 268 data points are shown. Two hundred and seven of the 286 gold particles marking FNR are non-randomly distributed with respect to gold particles marking GAPD B, and 155 of the 286 would be close enough to the gold particles marking GAPD B to indicate adjacent antigens, assuming complete linearity of the antigen-antibody complexes. (b) Plot of the negative log of 1 - fraction in the ordered list of measurements against the square of the distance between nearest neighbor gold particles when the particle sizes and relative antibody concentrations were reversed. There were 477 measurements. The first 464 data points are shown. Four hundred and nineteen of the 477 gold particles marking GAPD B are non-randomly distributed with respect to gold particles marking FNR, and 197 of the 477 would be close enough to the gold particles marking FNR to indicate adjacent antigens, assuming complete linearity of the antibody-antigen complexes.

Fig. 4

(a) Cartoon of the docked structures, based on PDB files 1jn0 (spinach GAPD) and 1fnb (spinach FNR). GAPD is shown in blue and FNR in red. The active sites are located on opposite sides of the area of interaction. (b) Interface residues, shown as red sticks for GAPD, and blue sticks for FNR. (c) Space filling model of the docked structures. (d) Cartoon of the docked GAPD and FNR structures overlaid on the structure of the maize FNR (pink), ferredoxin (light blue-green) complex in PDB file 1gaq. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 5

Basic residues connecting the FNR active site (in green, residues K116 and R117) and the GAPD active site (in yellow, residues R191, R194, and R195). NAPD binds into the active site cleft above and opposite R191. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 6

Conserved residues at putative interface in FNR in the model in Fig. 4. Number of sequences containing indicated residues in parentheses in left column. Completely redundant sequences within species not included. Conservative substitutions in gray. Only substitutions are shown.

Fig. 7

Residues corresponding to the residues in spinach chloroplast GAPD at the interface in the putative GAPD-FNR complex in the model in Fig. 4. Number of sequences containing indicated residues in parentheses in left column. Completely redundant sequences within species not included. Conservative substitutions in gray. Only substitutions are shown.

Fig. 8

(a) Cartoon showing the GAPD A₂B₂ tetramer (green, yellow, cyan, and magenta) docked to FNR (wheat). The structures are based on PDB files 1pkq and 1fnb. NADP in blue bound to GAPD. (b) Cartoon showing the GAPD A₂B₂ tetramer docked to an FNR dimer (wheat, orange). (c) Cartoon showing the GAPD A₂B₂ tetramer docked to the FNR (wheat), ferredoxin (pink) complex in PDB file 1ewy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 9

Conserved residues at putative interface with GAPD B subunit in FNR in the model in Fig. 8a. Number of sequences containing indicated residues in parentheses in left column. Completely redundant sequences within species not included. Conservative substitutions in gray. Only substitutions are shown.

Fig. 10

Conserved residues at putative interface with GAPD A subunit in FNR in the model in Fig. 8a. Number of sequences containing indicated residues in parentheses in left column. Completely redundant sequences within species not included. Conservative substitutions in gray. Only substitutions are shown.

Fig. 11

Residues corresponding to the residues in spinach chloroplast GAPD B at the interface with FNR in the putative GAPD-FNR complex in Fig. 8a. Number of sequences containing indicated residues in parentheses in left column. Completely redundant sequences within species not included. Conservative substitutions in gray. Only substitutions are shown.

Fig. 12

Residues corresponding to the residues in spinach chloroplast GAPD A at the interface with FNR in the putative GAPD-FNR complex in Fig. 8a. Number of sequences containing indicated residues in parentheses in left column. Completely redundant sequences within species not included. Conservative substitutions in gray. Only substitutions are shown.