The coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofluorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the first two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Interestingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838–6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes.

Ethylene induces enhanced differential growth in petioles of Arabidopsis (Arabidopsis thaliana), resulting in an upward movement of the leaf blades (hyponastic growth). The amplitude of this effect differs between accessions, with Columbia-0 (Col-0) showing a large response, while in Landsberg erecta (Ler), hyponastic growth is minimal. Abscisic acid (ABA) was found to act as an inhibitory factor of this response in both accessions, but the relationship between ethylene and ABA differed between the two; the ability of ABA to inhibit ethylene-induced hyponasty was significantly more pronounced in Col-0. Mutations in ABI1 or ABI3 induced a strong ethylene-regulated hyponastic growth in the less responsive accession Ler, while the response was abolished in the ABA-hypersensitive era1 in Col-0. Modifications in ABA levels altered petiole angles in the absence of applied ethylene, indicating that ABA influences petiole angles also independently from ethylene. A model is proposed whereby the negative effect of ABA on hyponastic growth is overcome by ethylene in Col-0 but not in Ler. However, when ABA signaling is artificially released in Ler, this regulatory mechanism is bypassed, resulting in a strong hyponastic response in this accession.