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Specificity of transmembrane protein palmitoylation in yeast.

González Montoro A, Chumpen Ramirez S, Quiroga R, Valdez Taubas J - PLoS ONE (2011)

Bottom Line: To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs) and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively.These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity.Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as expected for a highly specific enzymatic reaction.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs) and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as expected for a highly specific enzymatic reaction.

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Related in: MedlinePlus

Alignment of DHHC domains from selected model organisms and Groupsim SDP prediction results.Top three highest scoring positions in the GROUPSIM analysis are boxed. Residues are shaded according to conservation within orthologue groups. GROUPSIM scores and overall conservation for each position are shown below as box plots. The more comprehensive alignment used to run the GROUPSIM analysis is shown in Figure S1.
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pone-0016969-g004: Alignment of DHHC domains from selected model organisms and Groupsim SDP prediction results.Top three highest scoring positions in the GROUPSIM analysis are boxed. Residues are shaded according to conservation within orthologue groups. GROUPSIM scores and overall conservation for each position are shown below as box plots. The more comprehensive alignment used to run the GROUPSIM analysis is shown in Figure S1.

Mentions: Figure 4 displays an alignment of PATs DHHC domains from S. cerevisiae, Homo sapiens, Candida glabrata, Kluyveromyces lactis and Schizosaccharomyces pombe. Residues are shaded according to conservation within subgroups, and overall conservation is displayed in a bar graph below. GROUPSIM scores are displayed in bar graphs for each column, and the three top GROUPSIM columns are boxed, which correspond to A145, K148 and N168 in S. cerevisiae Swf1. The more comprehensive alignment found in File S1 was used to calculate the scores but Figure 4 can illustrate the conservation pattern of the predicted SDPs within their sequence context and also the identity of the residues for each PAT.


Specificity of transmembrane protein palmitoylation in yeast.

González Montoro A, Chumpen Ramirez S, Quiroga R, Valdez Taubas J - PLoS ONE (2011)

Alignment of DHHC domains from selected model organisms and Groupsim SDP prediction results.Top three highest scoring positions in the GROUPSIM analysis are boxed. Residues are shaded according to conservation within orthologue groups. GROUPSIM scores and overall conservation for each position are shown below as box plots. The more comprehensive alignment used to run the GROUPSIM analysis is shown in Figure S1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044718&req=5

pone-0016969-g004: Alignment of DHHC domains from selected model organisms and Groupsim SDP prediction results.Top three highest scoring positions in the GROUPSIM analysis are boxed. Residues are shaded according to conservation within orthologue groups. GROUPSIM scores and overall conservation for each position are shown below as box plots. The more comprehensive alignment used to run the GROUPSIM analysis is shown in Figure S1.
Mentions: Figure 4 displays an alignment of PATs DHHC domains from S. cerevisiae, Homo sapiens, Candida glabrata, Kluyveromyces lactis and Schizosaccharomyces pombe. Residues are shaded according to conservation within subgroups, and overall conservation is displayed in a bar graph below. GROUPSIM scores are displayed in bar graphs for each column, and the three top GROUPSIM columns are boxed, which correspond to A145, K148 and N168 in S. cerevisiae Swf1. The more comprehensive alignment found in File S1 was used to calculate the scores but Figure 4 can illustrate the conservation pattern of the predicted SDPs within their sequence context and also the identity of the residues for each PAT.

Bottom Line: To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs) and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively.These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity.Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as expected for a highly specific enzymatic reaction.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs) and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as expected for a highly specific enzymatic reaction.

Show MeSH
Related in: MedlinePlus