RESUMO
Membrane tethering is a fundamental process essential for the compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, are reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by trans-Rab assemblies on opposing membranes. Our findings support the novel concept that Rab-family proteins provide a bona fide membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane-tethering reactions.
Assuntos
Endossomos/metabolismo , Guanosina Trifosfato/metabolismo , Membranas Intracelulares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas rab de Ligação ao GTP/agonistas , Acilação , Endossomos/enzimologia , Histidina/química , Histidina/genética , Histidina/metabolismo , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/enzimologia , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/química , Miosina Tipo V/genética , Ácidos Oleicos/química , Ácidos Oleicos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Prenilação de Proteína , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Succinatos/química , Succinatos/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanócitos/enzimologia , Melanossomas/enzimologia , Proteínas rab de Ligação ao GTP/agonistas , Substituição de Aminoácidos , Animais , Linhagem Celular , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oxirredutases Intramoleculares/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Mutação , Oxirredutases/metabolismo , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Células 3T3 , Adipócitos/metabolismo , Animais , Transporte Biológico , Células COS , Linhagem Celular , Chlorocebus aethiops , Ativação Enzimática , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Glucose/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteínas rab de Ligação ao GTP/agonistasRESUMO
The human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood. Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl-terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaprost stimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time-dependent manner following cicaprost stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11(S25N) impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val(299)-Gln(320)) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.