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N-terminal binding domain of Galpha subunits: involvement of amino acids 11-14 of Galphao in membrane attachment.

ABSTRACT: Heterotrimeric guanine nucleotide binding proteins (G-proteins) transmit signals from membrane receptors to a variety of intracellular effectors. G-proteins reversibly associate with components of the signal transduction system, yet remain membrane attached throughout the cycle of activation. The Galpha subunits remain attached to the plasma membrane through a combination of factors that are only partially defined. We now demonstrate that amino acids within the N-terminal domain of Galpha subunits are involved in membrane binding. We used in vitro translation, a technique widely utilized to characterize functional aspects of G-proteins, and interactions with donor-acceptor membranes to demonstrate that amino acids 11-14 of Galphao contribute to membrane binding. The membrane binding of Galphao lacking amino acids 11-14 (D[11-14]) was significantly reduced at all membrane concentrations in comparison with wild-type Galphao. Several other N-terminal mutants of Galphao were characterized as controls, and these results indicate that differences in myristoylation, palmitoylation and betagamma interactions do not account for the reduced membrane binding of D[11-14]. Furthermore, when membrane attachment of Galphao and mutants was characterized in transiently transfected 35S-labelled and [3H]myristate-labelled COS cells, amino acids 11-14 contributed to membrane binding. These studies reveal that membrane binding of Galpha subunits occurs by a combination of factors that include lipids and amino acid sequences. These regions may provide novel sites for interaction with membrane components and allow additional modulation of signal transduction.

SUBMITTER: Busconi L

PROVIDER: S-EPMC1218301 | biostudies-other | 1997 Apr

REPOSITORIES: biostudies-other

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Project description:UnlabelledCS6 is a common colonization factor expressed by enterotoxigenic Escherichia coli It is a two-subunit protein consisting of CssA and CssB in an equal stoichiometry, assembled via the chaperone-usher pathway into an afimbrial, oligomeric assembly on the bacterial cell surface. A recent structural study has predicted the involvement of the N- and C-terminal regions of the CS6 subunits in its assembly. Here, we identified the functionally important residues in the N- and C-terminal regions of the CssA and CssB subunits during CS6 assembly by alanine scanning mutagenesis. Bacteria expressing mutant proteins were tested for binding with Caco-2 cells, and the results were analyzed with respect to the surface expression of mutant CS6. In this assay, many mutant proteins were not expressed on the surface while some showed reduced expression. It appeared that some, but not all, of the residues in both the N and C termini of CssA and CssB played an important role in the intermolecular interactions between these two structural subunits, as well as chaperone protein CssC. Our results demonstrated that T20, K25, F27, S36, Y143, and V147 were important for the stability of CssA, probably through interaction of CssC. We also found that I22, V29, and I33 of CssA and G154, Y156, L160, V162, F164, and Y165 of CssB were responsible for CssA-CssB intermolecular interactions. In addition, some of the hydrophobic residues in the C terminus of CssA and the N terminus of CssB were involved in the stabilization of higher-order complex formation. Overall, the results presented here might help in understanding the pathway used to assemble CS6 and predict its structure.ImportanceUnlike most other colonization factors, CS6 is nonfimbrial, and in a sense, its subunit composition and assembly are also unique. Here we report that both the N- and C-terminal amino acid residues of CssA and CssB play a critical role in the intermolecular interactions between them and assembly proteins. We found mainly that alternate hydrophobic residues present in these motifs are essential for the interaction between the structural subunits, as well as the chaperone and usher assembly proteins. Our results indicate the involvement of the side chains of identified amino acids in CS6 assembly. This study adds a step toward understanding the interactions between structural subunits of CS6 and assembly proteins during CS6 biogenesis.

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N-terminal binding domain of Galpha subunits: involvement of amino acids 11-14 of Galphao in membrane attachment.

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