RESUMEN
Membrane trafficking involves the collection of cargo into nascent transport vesicles that bud off from a donor compartment, translocate along cytoskeletal tracks, and then dock and fuse with their target membranes. Docking and fusion involve initial interaction at a distance (tethering), followed by a closer interaction that leads to pairing of vesicle SNARE proteins (v-SNAREs) with target membrane SNAREs (t-SNAREs), thereby catalyzing vesicle fusion. When tethering cannot take place, transport vesicles accumulate in the cytoplasm. Tethering is generally carried out by two broad classes of molecules: extended, coiled-coil proteins such as the so-called Golgin proteins, or multi-subunit complexes such as the Exocyst, COG or Dsl complexes. This review will focus on the most recent advances in terms of our understanding of the mechanism by which tethers carry out their roles, and new structural insights into tethering complex transactions.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Fusión de Membrana/fisiología , Proteínas SNARE/metabolismoRESUMEN
GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)-containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Aparato de Golgi/fisiología , Proteínas de la Membrana/metabolismo , Transporte de Proteínas/fisiología , Vesículas Transportadoras/fisiología , Animales , Bovinos , Línea Celular , Chlorocebus aethiops , Células Epiteliales/metabolismo , Aparato de Golgi/patología , Proteínas de la Matriz de Golgi , Células HeLa , Humanos , Proteínas de la Membrana/genética , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , ARN Interferente Pequeño/genética , Receptor IGF Tipo 2/metabolismo , Eliminación de Secuencia/fisiología , Transfección , Proteínas de Transporte Vesicular/metabolismo , Red trans-Golgi/fisiologíaRESUMEN
GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether.
Asunto(s)
Vesículas Citoplasmáticas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Red trans-Golgi/metabolismo , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Sitios de Unión , Aparato de Golgi/ultraestructura , Proteínas de la Matriz de Golgi , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Isoformas de Proteínas/genética , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rab/genética , Red trans-Golgi/ultraestructuraRESUMEN
Core binding factor (CBF) participates in specification of the hematopoietic stem cell and functions as a critical regulator of hematopoiesis. Translocation or point mutation of acute myeloid leukemia 1 (AML1)/RUNX1, which encodes the DNA-binding subunit of CBF, plays a central role in the pathogenesis of acute myeloid leukemia and myelodysplasia. We characterized the t(X;21)(p22.3;q22.1) in a patient with myelodysplasia that fuses AML1 in-frame to the novel partner gene FOG2/ZFPM2. The reciprocal gene fusions AML1-FOG2 and FOG2-AML1 are both expressed. AML1-FOG2, which fuses the DNA-binding domain of AML1 to most of FOG2, represses the transcriptional activity of both CBF and GATA1. AML1-FOG2 retains a motif that recruits the corepressor C-terminal binding protein (CtBP) and these proteins associate in a protein complex. These results suggest a central role for CtBP in AML1-FOG2 transcriptional repression and implicate coordinated disruption of the AML1 and GATAdevelopmental programs in the pathogenesis of myelodysplasia.