RESUMO
Protein S-acylation or palmitoylation is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of proteins through a thioester bond. Palmitoylation and palmitoyltransferases (PATs) have been linked to several types of cancers, diseases of the central nervous system and many infectious diseases where pathogens use the host cell machinery to palmitoylate their effectors. Despite the central importance of palmitoylation in cell physiology and disease, progress in the field has been hampered by the lack of potent-specific inhibitors of palmitoylation in general, and of individual PATs in particular. Herein, we present a yeast-based method for the high-throughput identification of small molecules that inhibit protein palmitoylation. The system is based on a reporter gene that responds to the acylation status of a palmitoylation substrate fused to a transcription factor. The method can be applied to heterologous PATs such as human DHHC20, mouse DHHC21 and also a PAT from the parasite Giardia lamblia. As a proof-of-principle, we screened for molecules that inhibit the palmitoylation of Yck2, a substrate of the yeast PAT Akr1. We tested 3200 compounds and were able to identify a candidate molecule, supporting the validity of our method.
Assuntos
Aciltransferases/antagonistas & inibidores , Lipoilação , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Giardia lamblia/efeitos dos fármacos , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
DHHC palmitoyltransferases (DHHC-PATs) are very peculiar in that, outside the DHHC domain, they are very divergent even across orthologs from closely related species. This represents a challenge for the bioinformatic analyses of these proteins. Sequence-based analyses and predictions require a valid sequence alignment, which for this family of proteins requires extensive manual curation and this is difficult to attain for the nonspecialist. Here we present a simple method for the in silico analysis of the sequence of a particular PAT, that would allow for the identification of important structural features and functional residues in a PAT or PAT family.
Assuntos
Acetiltransferases , Alinhamento de Sequência , Análise de Sequência de Proteína , Software , Acetiltransferases/química , Acetiltransferases/genética , Motivos de Aminoácidos , Biologia ComputacionalRESUMO
The molecular mechanisms underlying the ERα nuclear/cytoplasmic pool that modulates pituitary cell proliferation have been widely described, but it is still not clear how ERα is targeted to the plasma membrane. The aim of this study was to analyse ERα palmitoylation and the plasma membrane ERα (mERα) pool, and their participation in E2-triggered membrane-initiated signalling in normal and pituitary tumour cell growth. Cell cultures were prepared from anterior pituitaries of female Wistar rats and tumour GH3 cells, and treated with 10 nM of oestradiol (E2). The basal expression of ERα was higher in tumour GH3 than in normal pituitary cells. Full-length palmitoylated ERα was observed in normal and pituitary tumour cells, demonstrating that E2 stimulation increased both, ERα in plasma membrane and ERα and caveolin-1 interaction after short-term treatment. In addition, the Dhhc7 and Dhhc21 palmitoylases were negatively regulated after sustained stimulation of E2 for 3 h. Although the uptake of BrdU into the nucleus in normal pituitary cells was not modified by E2, a significant increase in the GH3 tumoural cell, as well as ERK1/2 activation, with this effect being mimicked by PPT, a selective antagonist of ERα. These proliferative effects were blocked by ICI 182780 and the global inhibitor of palmitoylation. These findings indicate that ERα palmitoylation modulated the mERα pool and consequently the ERK1/2 pathway, thereby contributing to pituitary tumour cell proliferation. These results suggest that the plasma membrane ERα pool might be related to the proliferative behaviour of prolactinoma and may be a marker of pituitary tumour growth.
Assuntos
Membrana Celular/metabolismo , Proliferação de Células , Receptor alfa de Estrogênio/metabolismo , Neoplasias Hipofisárias/metabolismo , Animais , Antineoplásicos Hormonais/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Estrogênios/farmacologia , Feminino , Fulvestranto/farmacologia , Expressão Gênica/efeitos dos fármacos , Lipoilação/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Ratos WistarRESUMO
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.
Assuntos
Endocitose , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Domínios Proteicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Protein S-acylation, also known as palmitoylation, consists of the addition of a lipid molecule to one or more cysteine residues through a thioester bond. This modification, which is widespread in eukaryotes, is thought to affect over 12% of the human proteome. S-acylation allows the reversible association of peripheral proteins with membranes or, in the case of integral membrane proteins, modulates their behavior within the plane of the membrane. This review focuses on the consequences of protein S-acylation on intracellular trafficking and membrane association. We summarize relevant information that illustrates how lipid modification of proteins plays an important role in dictating precise intracellular movements within cells by regulating membrane-cytosol exchange, through membrane microdomain segregation, or by modifying the flux of the proteins by means of vesicular or diffusional transport systems. Finally, we highlight some of the key open questions and major challenges in the field.
Assuntos
Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Acilação , Cisteína/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipoilação , Palmitatos/metabolismo , Transporte ProteicoRESUMO
Ganglioside glycosyltransferases (GGTs) are type II membrane proteins bearing a short N-terminal cytoplasmic tail, a transmembrane domain (TMD), and a lumenal catalytic domain. The expression and activity of these enzymes largely determine the quality of the glycolipids that decorate mammalian cell membranes. Many glycosyltransferases (GTs) are themselves glycosylated, and this is important for their proper localisation, but few if any other post-translational modifications of these proteins have been reported. Here, we show that the GGTs, ST3Gal-V, ST8Sia-I, and ß4GalNAcT-I are S-acylated at conserved cysteine residues located close to the cytoplasmic border of their TMDs. ST3Gal-II, a GT that sialylates glycolipids and glycoproteins, is also S-acylated at a conserved cysteine located in the N-terminal cytoplasmic tail. Many other GTs also possess cysteine residues in their cytoplasmic regions, suggesting that this modification occurs also on these GTs. S-acylation, commonly known as palmitoylation, is catalysed by a family of palmitoyltransferases (PATs) that are mostly localised at the Golgi complex but also at the endoplasmic reticulum (ER) and the plasma membrane. Using GT ER retention mutants, we found that S-acylation of ß4GalNAcT-I and ST3Gal-II takes place at different compartments, suggesting that these enzymes are not substrates of the same PAT. Finally, we found that cysteines that are the target of S-acylation on ß4GalNAcT-I and ST3Gal-II are involved in the formation of homodimers through disulphide bonds. We observed an increase in ST3Gal-II dimers in the presence of the PAT inhibitor 2-bromopalmitate, suggesting that GT homodimerisation may be regulating S-acylation.
Assuntos
N-Acetilgalactosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Sialiltransferases/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular , Sequência Conservada , Cricetulus , Cisteína/metabolismo , Dimerização , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Mutação , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Filogenia , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sialiltransferases/química , Sialiltransferases/genética , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
An accurate way to characterize the functional potential of a protein is to analyze recognized protein domains encoded by the genes in a given group. The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated trafficking. In this work, we investigate the function of the single ENTH-containing protein from the protist Giardia lamblia by testing its function in Saccharomyces cerevisiae. This protein, named GlENTHp (for G. lamblia ENTH protein), is involved in Giardia in endocytosis and in protein trafficking from the ER to the vacuoles, fulfilling the function of the ENTH proteins epsin and epsinR, respectively. There are two orthologs of epsin, Ent1p and Ent2p, and two orthologs of epsinR, Ent3p and Ent5p in S. cerevisiae. Although the expression of GlENTHp neither complemented growth in the ent1Δent2Δ mutant nor restored the GFP-Cps1 vacuolar trafficking defect in ent3Δent5Δ, it interfered with the normal function of Ent3/5 in the wild-type strain. The phenotype observed is linked to a defect in Cps1 localization and α-factor mating pheromone maturation. The finding that GlENTHp acts as dominant negative epsinR in yeast cells reinforces the phylogenetic data showing that GlENTHp belongs to the epsinR subfamily present in eukaryotes prior to their evolution into different taxa.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Evolução Molecular , Giardia lamblia/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Sequência de Aminoácidos , Animais , Genes Dominantes , Humanos , Organismos Geneticamente Modificados , Estrutura Terciária de Proteína/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de SequênciaRESUMO
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.
Assuntos
Aciltransferases/química , Lipoilação/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Aciltransferases/genética , Aciltransferases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de SequênciaRESUMO
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.
Assuntos
Regulação Fúngica da Expressão Gênica , Complexo de Golgi/metabolismo , Proteínas de Membrana/química , Proteínas Qa-SNARE/química , Proteínas Qc-SNARE/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cricetulus , Complexo de Golgi/ultraestrutura , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Aciltransferases/química , Proteínas Fúngicas/química , Yarrowia/enzimologia , Zinco/química , Aciltransferases/genética , Aciltransferases/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Complexos de Coordenação/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas de Inativação de Genes , Teste de Complementação Genética , Humanos , Lipoilação , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae , Análise de Sequência de Proteína , Homologia Estrutural de ProteínaRESUMO
BACKGROUND: Glucose induces H(+)-ATPase activation in Saccharomyces cerevisiae. Our previous study showed that (i) S. cerevisiae plasma membrane H(+)-ATPase forms a complex with acetylated tubulin (AcTub), resulting in inhibition of the enzyme activity; (ii) exogenous glucose addition results in the dissociation of the complex and recovery of the enzyme activity. METHODS: We used classic biochemical and molecular biology tools in order to identify the key components in the mechanism that leads to H(+)-ATPase activation after glucose treatment. RESULTS: We demonstrate that glucose-induced dissociation of the complex is due to pH-dependent activation of a protease that hydrolyzes membrane tubulin. Biochemical analysis identified a serine protease with a kDa of 35-40 and an isoelectric point between 8 and 9. Analysis of several knockout yeast strains led to the detection of Lpx1p as the serine protease responsible of tubulin proteolysis. When lpx1Δ cells were treated with glucose, tubulin was not degraded, the AcTub/H(+)-ATPase complex did not undergo dissociation, and H(+)-ATPase activation was significantly delayed. CONCLUSION: Our findings indicate that the mechanism of H(+)-ATPase activation by glucose involves a decrease in the cytosolic pH and consequent activation of a serine protease that hydrolyzes AcTub, accelerating the process of the AcTub/H(+)-ATPase complex dissociation and the activation of the enzyme. GENERAL SIGNIFICANCE: Our data sheds light into the mechanism by which acetylated tubulin dissociates from the yeast H(+)-ATPase, identifying a degradative step that remained unknown. This finding also proposes an indirect way to pharmacologically regulate yeast H(+)-ATPase activity and open the question about mechanistic similarities with other higher eukaryotes.
Assuntos
Adenosina Trifosfatases/metabolismo , Glucose/farmacologia , Proteínas de Membrana/metabolismo , Fosfolipases A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Serina Proteases/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação/efeitos dos fármacos , Adenosina Trifosfatases/genética , Membrana Celular/enzimologia , Membrana Celular/genética , Ativação Enzimática/efeitos dos fármacos , Proteínas de Membrana/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfolipases A/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina Proteases/genética , Tubulina (Proteína)/genéticaRESUMO
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.
Assuntos
Acetiltransferases/metabolismo , Lipoilação/fisiologia , Proteínas de Membrana/metabolismo , Leveduras/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/fisiologia , Aciltransferases/química , Aciltransferases/genética , Aciltransferases/metabolismo , Aciltransferases/fisiologia , Sequência de Aminoácidos , Domínio Catalítico/genética , Domínio Catalítico/fisiologia , Lipoilação/genética , Proteínas de Membrana/química , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/genética , Leveduras/genéticaRESUMO
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').
Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/genética , Motivos de Aminoácidos , Western Blotting , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
GalT2 (UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase) is a Golgi-resident type II membrane protein that participates in the synthesis of glycosphingolipids. The molecular determinants for traffic and localization of this and other glycosyltransferases are still poorly characterized. Considering the possibility that interactions with other proteins may influence these processes, in the present study we carried out a yeast two-hybrid screening using elements of the N-terminal domain of GalT2 as bait. In this screening, we identified calsenilin and its close homologue CALP (calsenilin-like protein), both members of the recoverin-NCS (neuronal calcium sensor) family of calcium-binding proteins. In vitro, GalT2 binds to immobilized recombinant CALP, and CALP binds to immobilized peptides with the GalT2 cytoplasmic tail sequence. GalT2 and calsenilin interact physically when co-expressed in CHO (Chinese-hamster ovary)-K1 cells. The expression of CALP or calsenilin affect Golgi localization of GalT2, and of two other glycosyltransferases, SialT2 (CMP-NeuAc:GM3 sialyltransferase) and GalNAcT (UDP-GalNAc:lactosylceramide/GM3/GD3 beta1-4 N-acetylgalactosaminyltransferase), by redistributing them from the Golgi to the ER (endoplasmic reticulum), whereas the localization of the VSV-G (G-protein of the vesicular stomatitis virus) or the Golgin GM130 was essentially unaffected. Conversely, the expression of GalT2 affects the localization of calsenilin and CALP by shifting a fraction of the molecules from being mostly diffuse in the cytosol, to clustered structures in the perinuclear region. These combined in vivo and in vitro results suggest that CALP and calsenilin are involved in the trafficking of Golgi glycosyltransferases.
Assuntos
Galactosiltransferases/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Retículo Endoplasmático/metabolismo , Galactosiltransferases/química , Complexo de Golgi/metabolismo , Humanos , Proteínas Interatuantes com Canais de Kv/fisiologia , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Técnicas do Sistema de Duplo-HíbridoRESUMO
The Aspergillus nidulans UapC protein is a high-affinity, moderate-capacity, uric acid-xanthine transporter, which also displays a low transport capacity for hypoxanthine, adenine, and guanine. It has been previously shown that a functional UapC-GFP fusion protein localises at the plasma membrane. Here, we demonstrate that ammonium, a preferred nitrogen source, dramatically changes the subcellular distribution of UapC. After addition of ammonium, UapC-GFP is removed from the plasma membrane and is concentrated into the vacuolar compartment. A chimeric gene construct in which an inducible promoter, insensitive to nitrogen repression, drives the expression of UapC-GFP, allowed us to demonstrate that the ammonium-dependent redistribution of UapC can be dissociated from the transcriptional repression of the gene. These results provide further support for the occurrence of endocytosis and the lysosomal-endosomal function of the vacuolar compartment in A. nidulans.