RESUMEN
SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.
Asunto(s)
Dominio Catalítico , Mutagénesis Sitio-Dirigida , Esfingolípidos/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Dominios Proteicos , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, sphingomyelin synthase-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmatic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with glutamic acid permitting SMS-catalyzed CPE production and aspartic acid confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Ingeniería de Proteínas , Esfingomielinas/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Membrana Celular/enzimología , Membrana Celular/metabolismo , Sistema Libre de Células , Química Clic , Retículo Endoplásmico/enzimología , Aparato de Golgi/enzimología , Células HeLa , Humanos , Proteínas de la Membrana/química , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/química , Esfingomielinas/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/químicaRESUMEN
Maturation of organelles in the endolysosomal pathway requires exchange of the early endosomal GTPase Rab5/Vps21 for the late endosomal Rab7/Ypt7. The Rab exchange depends on the guanine nucleotide exchange factor activity of the Mon1-Ccz1 heterodimer for Ypt7. Here we investigate vacuole binding and recycling of Mon1-Ccz1. We find that Mon1-Ccz1 is absent on vacuoles lacking the phosphatidic acid phosphatase Pah1, which also lack Ypt7, the phosphatidylinositol 3-kinase Vps34, and the lipid phosphatidylinositol 3-phosphate (PI3P). Interaction of Mon1-Ccz1 with wild-type vacuoles requires PI3P, as shown in competition experiments. We also find that Mon1 is released from vacuoles during the fusion reaction and its release requires its phosphorylation by the type 1 casein kinase Yck3. In contrast, Mon1 is retained on vacuoles lacking Yck3 or when Mon1 phosphorylation sites are mutated. Phosphorylation and release of Mon1 is restored with addition of recombinant Yck3. Together the results show that Mon1 is recruited to endosomes and vacuoles by PI3P and, likely after activating Ypt7, is phosphorylated and released from vacuoles for recycling.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Quinasa de la Caseína I/genética , Fosfatidilinositol 3-Quinasas Clase III/genética , Endosomas/metabolismo , Fosfatidato Fosfatasa/genética , Fosfatos de Fosfatidilinositol/genética , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab5 , Proteínas de Unión a GTP rab7RESUMEN
To function in fusion and signaling, Rab GTPases need to be converted into their active GTP form. We previously identified the conserved Mon1-Ccz1 complex as the guanine nucleotide exchange factor (GEF) of the yeast Rab7 GTPase Ypt7. To address the possible GEF mechanism, we generated a homology model of the predicted longin domains of Mon1 and Ccz1 using the Rab-binding surface of the TRAPP complex as a template. On the basis of this, we identified mutations in both yeast Mon1 and Ccz1 that block Ypt7 activation, without affecting heterodimer formation and intracellular localization of Mon1 and Ccz1 at endosomes. Strikingly, the activity of the isolated Mon1-Ccz1 complex for Ypt7 is highly stimulated on membranes, and is promoted by the same anionic phospholipids such as phosphatidylinositol-3-phosphate (PI3P), which also support membrane association of the GEF complex. Our data imply that the GEF activity of the Mon1-Ccz1 complex towards Rab7/Ypt7 requires the interface formed by their longin domains and profits strongly from its association with the organelle surface.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Membranas Intracelulares/enzimología , Fosfatos de Fosfatidilinositol/química , Fosfatidilserinas/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Vacuolas/metabolismo , Vacuolas/ultraestructura , Proteínas de Transporte Vesicular/química , Proteínas de Unión al GTP rab/químicaRESUMEN
Rab GTPases coordinate membrane fusion reactions [1]. Rab-GDP requires a guanine nucleotide exchange factor (GEF) for its conversion to the active GTP form. It then binds to effectors such as multimeric tethering complexes and supports fusion [2]. GTPase-activating proteins (GAPs) promote GTP hydrolysis to inactivate the Rab. GEFs are thus critical activators of fusion reactions [3, 4]. The Rab GEF family is diverse, ranging from multimeric complexes [5] to monomeric GEFs [6-9]. At the late endosome, Rab7 activation is critical for endosomal maturation. The yeast Rab7 homolog Ypt7 binds to the homotypic fusion and protein sorting (HOPS) complex [10, 11]. Its subunit Vps39/Vam6 has been proposed as a GEF for Ypt7 [12] and the Rag GTPase Gtr1 [13], but other genetic evidence has implicated the endosomal protein Ccz1 as a GEF for Ypt7 [14]. Ccz1 and its binding partner Mon1 have been linked to endosomal transport and maturation [15-20]. We now provide evidence that the dimeric Mon1-Ccz1 complex is the Rab7/Ypt7 GEF. The Mon1-Ccz1 complex, but neither protein alone, counteracts GAP function in vivo, rescues in vitro fusion of vacuoles carrying Ypt7-GDP, and promotes nucleotide exchange on Ypt7 independently of Vps39/HOPS. Our data indicate that the Mon1-Ccz1 complex triggers endosomal maturation by activating Ypt7 on late endosomes.
Asunto(s)
Endosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Complejos Multiproteicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/genética , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
Within the endomembrane system of eukaryotic cells, multisubunit tethering complexes together with their corresponding Rab-GTPases coordinate vesicle tethering and fusion. Here, we present evidence that two homologous hexameric tethering complexes, the endosomal CORVET (Class C core vacuole/endosome transport) and the vacuolar HOPS (homotypic vacuole fusion and protein sorting) complex, have similar subunit topologies. Both complexes contain two Rab-binding proteins at one end, and the Sec1/Munc18-like Vps33 at the opposite side, suggesting a model on membrane bridging via Rab-GTP and SNARE binding. In agreement, HOPS activity can be reconstituted using purified subcomplexes containing the Rab and Vps33 module, but requires all six subunits for activity. At the center of HOPS and CORVET, the class C proteins Vps11 and Vps18 connect the two parts, and Vps11 binds both HOPS Vps39 and CORVET Vps3 via the same binding site. As HOPS Vps39 is also found at endosomes, our data thus suggest that these tethering complexes follow defined but distinct assembly pathways, and may undergo transition by simple subunit interchange.