In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy

Abstract

The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes.

Figure 1.

Expression in Arabidopsis protoplasts of CDC48A, CDC48A^A1A2, CDC48A^Ndel, and CDC48A^N-D1 mutants all tagged to YFP. YFP is replaced by CrFP. In A the various mutants are shown schematically. Lysates of transfected cells were submitted to SDS-PAGE in B, to native PAGE in C, and probed with anti-YFP anti-serum. The expected sizes of the denatured proteins are 125 kD for full-length (FL) CDC48A- and CDC48A^A1A2-YFP, 96 kD for CDC48A^Ndel-YFP, and 77 kD for CDC48A^N-D1-YFP. Nondenatured hexameric CDC48A-YFP is expected to be between 650 and 750 kD depending on the number of YFP tags.

Figure 2.

Localizations of CDC48A- or CDC48A^N-D1- and CDC48A^Ndel-YFP. CDC48C-CrFP was used as a nuclear marker (B, E, H, and K). Images were taken at the plane of the nucleus and the merged picture shows the combination of both images together with the chloroplasts in red (C, F, I, and L). CDC48A-YFP localizes to the cytoplasm and the nucleus (compare position of arrowhead in A with image B). The localization at the PM is not visible in this focal plane. CDC48A^N-D1 does localize to the cytoplasm but not to the nucleus (compare the arrowhead in image D with E). CDC48A^Ndel does localize to the cytoplasm and to the nucleus (compare the arrowhead in G with H). The localization of CDC48A^A1A2 (J) is comparable with that of CDC48A (A).

Figure 3.

Models of the expected hexamers of CDC48A after coexpression of two different protomers. A, Monomeric CDC48A. B, CDC48A fused with CrFP and with YFP (both at the C terminus) are combined and depicted as a hexamer. For simplicity only four fluorophores are drawn at adjacent positions at the C terminus, but in fact these can be randomly positioned at each C domain. C, YFP fused to the N terminus and CrFP to the C terminus. D, YFP and CrFP fused to the N terminus. E and F, CDC48A^N-D1 and CDC48A^Ndel, each depicted as a monomer.

Figure 4.

Interactions between CDC48A-tagged protomers based on FRET measured by FLIM. A, Intensity images of the PM of part of a protoplast expressing the donor molecule CDC48-CrFP alone; B, the false-color or lifetime image. A long lifetime, giving a dark blue color, means no interaction; a reduction in donor lifetime, generating a shift toward orange, means interaction. The combination of CDC48A-CrFP and SERK1-YFP shows a reduction in lifetime at specific regions at the PM (C and D plus inserts). A combination of CDC48A-CrFP and CDC48A-YFP proteins shows a reduction in lifetime at the PM in the same regions where SERK1 and CDC48A interact (F plus insert) and in the nucleus (H). In the left top corners, intensity images are shown. A combination of CDC48A-CrFP and YFP-CDC48A at the PM is shown in I and in the nucleus in J. K and L, Images of CrFP-CDC48A alone at the PM (K) and in combination with YFP-CDC48A (L), showing no reduction in lifetime. M and N, Lifetime images of the CDC48A^N-D1-CrFP protein as the donor (M) and the combination with a CDC48A^N-D1-YFP protein (N). O and P, Lifetime images of the CrFP-CDC48C donor alone (O) and in combination with YFP-CDC48C (P). Q and R, Combination of CDC48C-CrFP with YFP-CDC48C (Q) or with CDC48C-YFP (R) shows a reduction in lifetime.

Figure 5.

Representative lifetime distribution histograms measured with FLIM at the PM of Arabidopsis protoplasts. A, Histogram of the CDC48A donor alone. B, Histogram of a mixture of CDC48A tagged to CrFP and YFP at the C terminus, which shifted to lower lifetime values due to interaction. C, Histogram of a mixture of CDC48A tagged to CrFP and YFP at the N terminus. No significant shift to lower lifetime values is visible.

Figure 6.

In vitro trans-phosphorylation of CDC48A by SERK1 kinase protein. A, Left, Phosphor image of CDC48A trans-phosphorylated by the SERK1 kinase domain. GST-CDC48A, GST alone, or water was incubated with the SERK1 kinase domain (SK1-KD) in a buffer containing [γ³²-P]ATP (lane 1, 2, and 3, respectively). Two control experiments were performed without SK1-KD. SK1-KD is autophosphorylated and trans-phosphorylates CDC48A (arrowhead, lane 1). A, Right, Equal loading and size control of GST-CDC48A, GST, and SK1-KD protein. B, Peptide coverage of CDC48A in two reactions containing CDC48A alone or CDC48A plus SK1-KD was 66% and 68%, respectively (colored in gray). Ser-41 of CDC48A was phosphorylated by SK1-KD.

Figure 7.

Representative autocorrelation curves from CDC48A-YFP (squares), CDC48A^Ndel-YFP (triangles), and CDC48A^N-D1-YFP (lines), compared to the curve for free YFP (lowest curve: diamonds) in Arabidopsis protoplasts. The autocorrelation curves were fitted with a one-component model. On the y axis the autocorrelation function G(τ) is depicted. The correlation time τ (ms) is depicted on the x axis in log scale.