RESUMEN

Endocytosis is crucial for all cells as it allows them to incorporate material from the extracellular space and control the availability of transmembrane proteins at the plasma membrane. In yeast, endocytosis followed by recycling to the plasma membrane results in a polarised distribution of membrane proteins by a kinetic mechanism. Here, we report that increasing the volume of residues that constitute the exoplasmic half of the transmembrane domain (TMD) in the yeast SNARE Sso1, a type II membrane protein, results in its polarised distribution at the plasma membrane. Expression of this chimera in strains affected in either endocytosis or recycling revealed that this polarisation is achieved by endocytic cycling. A bioinformatics search of the Saccharomyces cerevisiae proteome identified several proteins with high-volume exoplasmic hemi-TMDs. Our experiments indicate that TMDs from these proteins can confer a polarised distribution to the Sso1 cytoplasmic domain, indicating that the shape of the TMD can act as a novel endocytosis and polarity signal in yeast. Additionally, a high-volume exoplasmic hemi-TMD can act as an endocytosis signal in a mammalian cell line.

Asunto(s)

Endocitosis , Secuencia de Aminoácidos , Animales , Células CHO , Cricetulus , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Dominios Proteicos , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismo

RESUMEN

Protein S-acyltransferases, also known as palmitoyltransferases (PATs), are characterized by the presence of a 50-amino acid domain called the DHHC domain. Within this domain, these four amino acids constitute a highly conserved motif. It has been proposed that the palmitoylation reaction occurs through a palmitoyl-PAT covalent intermediate that involves the conserved cysteine in the DHHC motif. Mutation of this cysteine results in lack of function for several PATs, and DHHA or DHHS mutants are used regularly as catalytically inactive controls. In a genetic screen to isolate loss-of-function mutations in the yeast PAT Swf1, we isolated an allele encoding a Swf1 DHHR mutant. Overexpression of this mutant is able to partially complement a swf1Δ strain and to acylate the Swf1 substrates Tlg1, Syn8, and Snc1. Overexpression of the palmitoyltransferase Pfa4 DHHA or DHHR mutants also results in palmitoylation of its substrate Chs3. We also investigated the role of the first histidine of the DHHC motif. A Swf1 DQHC mutant is also partially active but a DQHR is not. Finally, we show that Swf1 substrates are differentially modified by both DHHR and DQHC Swf1 mutants. We propose that, in the absence of the canonical mechanism, alternative suboptimal mechanisms take place that are more dependent on the reactivity of the acceptor protein. These results also imply that caution must be exercised when proposing non-canonical roles for PATs on the basis of considering DHHC mutants as catalytically inactive and, more generally, contribute to an understanding of the mechanism of protein palmitoylation.

Asunto(s)

Aciltransferasas/química , Lipoilación/fisiología , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia

RESUMEN

It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the Golgi SNARE protein Sft1 and the plasma membrane SNARE protein Sso1 from Saccharomyces cerevisiae as model proteins, we modified the length of their TMDs and the volume of their exoplasmic hemi-TMD, and determined their subcellular localization both in yeast and mammalian cells. We found that short TMDs with high-volume exoplasmic hemi-TMDs confer Golgi membrane residence, whereas TMDs with low-volume exoplasmic hemi-TMDs, either short or long, confer plasma membrane residence to these proteins. Results indicate that the shape of the exoplasmic hemi-TMD, in addition to the length of the entire TMD, determine retention in the Golgi or exit to the plasma membrane of Type II membrane proteins.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Aparato de Golgi/metabolismo , Proteínas de la Membrana/química , Proteínas Qa-SNARE/química , Proteínas Qc-SNARE/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Cricetulus , Aparato de Golgi/ultraestructura , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMEN

S-acylation, commonly known as palmitoylation, is a widespread post-translational modification of proteins that consists of the thioesterification of one or more cysteine residues with fatty acids. This modification is catalysed by a family of PATs (palmitoyltransferases), characterized by the presence of a 50-residue long DHHC-CRD (Asp-His-His-Cys cysteine-rich domain). To gain knowledge on the structure-function relationships of these proteins, we carried out a random-mutagenesis assay designed to uncover essential amino acids in Swf1, the yeast PAT responsible for the palmitoylation of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins. We identified 21 novel loss-of-function mutations, which are mostly localized within the DHHC-CRD. Modelling of the tertiary structure of the Swf1 DHHC domain suggests that it could fold as a zinc-finger domain, co-ordinating two zinc atoms in a CCHC arrangement. All residues predicted to be involved in the co-ordination of zinc were found to be essential for Swf1 function in the screen. Moreover, these mutations result in unstable proteins, in agreement with a structural role for these zinc fingers. The conservation of amino acids predicted to form each zinc-binding pocket suggests a shared function, as the selective pressure to maintain them is lost upon mutation of one of them. A Swf1 orthologue that lacks one of the zinc-binding pockets is able to complement a yeast swf1∆ strain, possibly because a similar fold can be stabilized by hydrogen bonds instead of zinc co-ordination. Finally, we show directly that recombinant Swf1 DHHC-CRD is able to bind zinc. Sequence analyses of DHHC domains allowed us to present models of the zinc-binding properties for all PATs.

Asunto(s)

Aciltransferasas/química , Proteínas Fúngicas/química , Yarrowia/enzimología , Zinc/química , Aciltransferasas/genética , Aciltransferasas/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Complejos de Coordinación/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Humanos , Lipoilación , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae , Análisis de Secuencia de Proteína , Homología Estructural de Proteína

RESUMEN

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.

Asunto(s)

Acetiltransferasas/metabolismo , Lipoilación/fisiología , Proteínas de la Membrana/metabolismo , Levaduras/metabolismo , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/fisiología , Aciltransferasas/química , Aciltransferasas/genética , Aciltransferasas/metabolismo , Aciltransferasas/fisiología , Secuencia de Aminoácidos , Dominio Catalítico/genética , Dominio Catalítico/fisiología , Lipoilación/genética , Proteínas de la Membrana/química , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Homología de Secuencia de Aminoácido , Especificidad por Sustrato/genética , Levaduras/genética

RESUMEN

S-acylation (commonly known as palmitoylation) is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of a protein through a thioester bond. This modification is predominantly mediated by a family of proteins referred to as PATs (palmitoyltransferases). Most PATs are polytopic membrane proteins, with four to six transmembrane domains, a conserved DHHC motif and variable C-and N-terminal regions, that are probably responsible for conferring localization and substrate specificity. There is very little additional information on the structure-function relationship of PATs. Swf1 and Pfa3 are yeast members of the DHHC family of proteins. Swf1 is responsible for the S-acylation of several transmembrane SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) and other integral membrane proteins. Pfa3 is required for the palmitoylation of Vac8, a protein involved in vacuolar fusion. In the present study we describe a novel 16-amino-acid motif present at the cytosolic C-terminus of PATs, that is required for Swf1 and Pfa3 function in vivo. Within this motif, we have identified a single residue in Swf1, Tyr323, as essential for function, and this is correlated with lack of palmitoylation of Tlg1, a SNARE that is a substrate of Swf1. The equivalent mutation in Pfa3 also affects its function. These mutations are the first phenotype-affecting mutations uncovered that do not lie within the DHHC domain, for these or any other PATs. The motif is conserved in 70% of PATs from all eukaryotic organisms analysed, and may have once been present in all PATs. We have named this motif PaCCT ('Palmitoyltransferase Conserved C-Terminus').

Asunto(s)

Aciltransferasas/química , Aciltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferasas/genética , Secuencias de Aminoácidos , Western Blotting , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-Actividad