Heterodimeric interactions among the 1-amino-cyclopropane-1-carboxylate synthase polypeptides encoded by the Arabidopsis gene family

Abstract

The pyridoxal phosphate-dependent enzyme, 1-aminocyclopropane-1-carboxylate synthase (ACS; EC 4.4.1.14), catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. The Arabidopsis genome encodes nine ACS polypeptides that form eight functional (ACS2, ACS4-9, ACS11) and one nonfunctional (ACS1) homodimers. Because the enzyme is a homodimer with shared active sites, the question arises whether the various polypeptides can form functional heterodimers. Intermolecular complementation experiments in Escherichia coli by coexpressing the K278A and Y92A mutants of different polypeptides show that all of them have the capacity to heterodimerize. However, functional heterodimers are formed only among gene family members that belong to one or the other of the two phylogenetic branches. ACS7 is an exception to this rule, which forms functional heterodimers with some members of both branches when it provides the wt K278 residue. ACS1, the nonfunctional polypeptide as a homodimer, can also form functional heterodimers with members of its phylogenetic branch when its partners provide the wt K278 residue. The ACS gene family products can potentially form 45 homo- and heterodimers of which 25 are functional. Bimolecular fluorescence complementation and biochemical coaffinity purification assays show that the inactivity of certain heterodimers is not due to the absence of heterodimerization but rather to structural restraint(s) that prevents the shared active sites from being functional. We propose that functional heterodimerization enhances the isozyme diversity of the ACS gene family and provides physiological versatility by being able to operate in a broad gradient of S-adenosylmethionine concentration in various cells/tissues during plant growth and development. Nonfunctional heterodimerization may also play a regulatory role during the plant life cycle.

Fig. 1.

The strategy for determining heterodimeric interactions among the various polypeptides encoded by the Arabidopsis ACS gene family members. (A) The principle of the method: schematic presentation of an active heterodimer formation by coexpressing the K278A and Y92A mutants of two ACS polypeptides X and Z, respectively. (B) The construct: a pQE double-gene construct used to coexpress the K278A and Y92A mutants of various ACSs as His- and T7-tagged proteins with the T5 promoter. (C) The E. coli strain JAde6. The biosynthetic pathway of isoleucine in the E. coli auxotroph JHM544 is shown (shaded blue). The pathway was engineered to allow Ile prototrophy by integrating ACC deaminase into the genome, yielding E. coli JAde6 (19). The strain can metabolize ACC synthesized when transformed with a functional ACS heterodimer. MTA, methylthioadenosine.

Fig. 2.

Growth of E. coli JAde6 expressing mutant ACSs. (A and B) Growth of JAde6 on rich (LB) and minimum (M9) media coexpressing the K278A and Y92A mutants of various ACSs. (C–F) Schematic presentation of the results in B regarding the formation of active and inactive heterodimers in relation to their phylogenetic location. (C) Functional homodimerization by coexpressing K278A and Y92A mutants of ACSs. (D) Functional heterodimerization among the various ACSs depending on their phylogenetic location. (E) Functional heterodimerization between the Y92 mutants of ACS7 and some members of both phylogenetic branches. (F) Functional heterodimerization between the wt ACS1 and the Y92A mutants of ACS2 and ACS6, respectively. AspAT, aspartate aminotransferase; AlaAT, alanine aminotransferase.

Fig. 3.

Determination of protein–protein interactions in active and inactive ACS heterodimers. (A) Schematic presentation of the biochemical coaffinity purification assay. Dimers of His- and T7-tagged K278A and Y92A mutants of ACS were purified by Ni-NTA affinity chromatography as described in Supporting Text and analyzed by immunoblotting with His and T7 antibodies. (B) ACS4/ACS6 heterodimers. Lane 1, H-ACS4K278A; lane 2, T-ACS4Y92A; lane 3, H-ACS6K278A; lane 4, T-ACS6Y92A; lane 5, H-ACS4K278A/T7-ACS4Y92A; lane 6, H-ACS6K278A/T7-ACS6Y92A; lane 7, H-ACS6K278A/T7-ACS4Y92A. (C) ACS4/ACS8 heterodimers. Lane 1, H-ACS4K278A; lane 2, T-ACS4Y92A; lane 3, H-ACS8K278A; lane 4, T-ACS8Y92A; lane 5, H-ACS4K278A/T-ACS4Y92A; lane 6, H-ACS8K278A/T-ACS8Y92A; lane 7, H-ACS8K278A/T-ACS4Y92A. (D) ACS4/ACS7 heterodimers. Lane 1, H-ACS4K278A; lane 2, T-ACS4Y92A; lane 3, H-ACS7K278A; lane 4, T-ACS7Y92A; lane 5, H-ACS4K278A/T-ACS4Y92A; lane 6, His-ACS7K278A/T-ACS7Y92A; lane 7, H-ACS7K278A/T-ACS4Y92A; lane 8, H-ACS4K278A/T-ACS7Y92A. (E) ACS1/ACS6 heterodimers. Lane 1, H-ACS1K278A; lane 2, T-ACS1Y92A; lane 3, H-ACS6K278A; lane 4, T-ACS6Y92A; lane 5, H-ACS1K278A/T-ACS1Y92A; lane 6, H-ACS6K278A/T-ACS6Y92A; lane 7, H-ACS6K278A/T-ACS1Y92A; lane 8, H-ACS1K278A/T-ACS6Y92A. The upper two rows in B–E are immunoblots with total E. coli extracts before Ni-NTA affinity chromatography. The lower two rows in B–E are immunoblots after Ni-NTA affinity chromatography. V, a protein sample of the E. coli DH5α with the vector alone; A and I, active and inactive homo- or heterodimers, respectively; H and T, His- and T7-tags, respectively

Fig. 4.

BiFC. (A) Principle of the BiFC method for detecting bFos:bJun heterodimerization and ACS6:ACS6 homodimerization. The N and C termini of YFP marked in red and green, respectively, were fused to the C terminus of bJun and bFos, respectively, or to the N terminus of ACS6. (B) The gene constructs used for transforming E. coli and visualization of protein–protein interaction by YFP fluorescence imaging as described in Supporting Text.

Fig. 5.

Detection of ACS heterodimer formation by BiFC. (A–D) Detection of heterodimerization between the K278A and Y92A mutants of ACS6 and ACS4 that yields inactive heterodimers. (A) The gene constructs used for detecting ACS4/ACS6 heterodimerization are numbered from 1 to 7. (B) ACS activity of E. coli strain DH5α transformed with the constructs shown above. (C) Western blot analysis of crude E. coli extracts expressing the gene constructs shown in A with use of His and T7 monoclonal antibodies. The analysis also shows immunoblotting of E. coli extracts expressing non-YN or -YC fusions of ACS4 and ACS6 K278A and Y92A mutants, respectively. (D) Visualization of protein–protein interaction by YFP imaging as described in Supporting Text. (E–H) Detection of heterodimerization between the K278A and Y92A mutants of ACS4 and ACS8 that yields active heterodimers. F, G, and H are the same as in B, C, and D, except they correspond to ACS4/ACS8 heterodimer.