Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy of snf1⌬ strains. We identified mutations in ACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the  subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show that snf1⌬ strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying the ACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression of ACC1, suppresses the inositol auxotrophy of snf1⌬ strains. Together, these findings indicate that the inositol auxotrophy of snf1⌬ strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important for INO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.
MARCKS-related protein (MRP) isT he proteins of the myristoylated alanine-rich C kinase substrate (MARCKS) family are protein kinase C substrates that have been proposed to regulate the actin cytoskeleton (1). The family comprises two members: MARCKS itself is a ubiquitous 32-kDa protein, whereas MARCKS-related protein (MRP, also called MacMARCKS) is a 22-kDa protein expressed mainly in brain and reproductive tissues (2). MARCKS proteins share two conserved segments, namely the myristoylated N terminus and a central highly basic 24-to 25-residue-long segment, the effector domain, also called the ''phosphorylation site domain'' (1, 3). In vitro, MARCKS and MRP bind to calmodulin (CaM) with high affinity (K d Ϸ 5 nM) (4, 5). Although direct proof for an interaction between MARCKS proteins and CaM has so far not been obtained in cells, indirect evidence suggests that MARCKS proteins mediate crosstalk between the protein kinase C-and CaM-signal transduction pathways (for reviews, see refs. 6 and 7).Two segments in MARCKS proteins are of interest with respect to their interactions with CaM.(i) The Effector Domain. The structure of the effector domain of MARCKS proteins has been the subject of several reports. Fig. 1 shows the amino acid sequence of the effector domain of MRP. This segment is highly basic (12 residues of 24 are lysines or arginines), but it also contains most of the large hydrophobic residues present in the intact protein (four phenylalanines and two leucines of seven). Although sequence analysis has previously led to the conclusion that this segment forms an ␣-helix (1), circular dichroic studies with peptides (8) and proteins (5, 9) suggest an extended structure for this segment. In the absence of information on the tertiary structure of MARCKS proteins, three different topologies can be proposed for the effector domain. The effector domain could be completely buried in a hydrophobic core (model A), which is unlikely because of the high content of charged amino acid residues. The effector domain could be on the surface of MARCKS proteins and partially exposed to water (model B). This model fits the proposed amphipathic structure of the effector domain (basic͞hydrophobic residues). Finally, the effector domain could be completely exposed to water and either act as a hinge between the C-and N-terminal domains of MARCKS proteins (model C1) or form an exposed loop on the surface of the protein (model C2). The observation that MARCKS proteins are elongated molecules (5, 10) supports model C1. However, the presence of a proline at position 96 in the middle of the effector domain of MRP could potentially induce the formation of a kink or a turn (11), favoring model C2.(ii) The Myristoylated N Terminus. Covalent modification of proteins with lipids, such as myristoylation in the case of MARCKS proteins, allows the interactions of these proteins with membranes (12). Myristoylation could also mediate protein-protein interactions. However, most proteins modified with lipids interact with other proteins on the ...
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