RESUMO
Dimerization of the small GTPase Arf is prerequisite for the scission of COPI-coated transport vesicles. Here, we quantify the monomer/dimer equilibrium of Arf within the membrane and show that after membrane scission, Arf dimers are restricted to donor membranes. By hydrogen exchange mass spectrometry, we define the interface of activated dimeric Arf within its switch II region. Single amino acid exchanges in this region reduce the propensity of Arf to dimerize. We suggest a mechanism of membrane scission by which the dimeric form of Arf is segregated to the donor membrane. Our data are consistent with the previously reported absence of dimerized Arf in COPI vesicles and could explain the presence of one single scar-like noncoated region in each COPI vesicle.
Assuntos
Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Membrana Celular/metabolismo , Multimerização Proteica , Sítios de Ligação , Humanos , Bicamadas Lipídicas/metabolismo , Modelos MolecularesRESUMO
In the formation of COPI vesicles, interactions take place between the coat protein coatomer and membrane proteins: either cargo proteins for retrieval to the endoplasmic reticulum (ER) or proteins that cycle between the ER and the Golgi. While the binding sites on coatomer for ER residents have been characterized, how cycling proteins bind to the COPI coat is still not clear. In order to understand at a molecular level the mechanism of uptake of such proteins, we have investigated the binding to coatomer of p24 proteins as examples of cycling proteins as well as that of ER-resident cargos. The p24 proteins required dimerization to interact with coatomer at two independent binding sites in gamma-COP. In contrast, ER-resident cargos bind to coatomer as monomers and to sites other than gamma-COP. The COPI coat therefore discriminates between p24 proteins and ER-resident proteins by differential binding involving distinct subunits.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Transporte/metabolismo , Proteína Coatomer/metabolismo , Retículo Endoplasmático/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteína Coatomer/química , Proteína Coatomer/genética , Dimerização , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de ProteínaRESUMO
The Wiskott-Aldrich syndrome protein family member N-WASP is a key integrator of the multiple signalling pathways that regulate actin polymerization via the Arp2/3 complex. Our previous studies have shown that N-WASP is required for the actin-based motility of vaccinia virus and is recruited via Nck and WIP. We now show that Grb2 is an additional component of the vaccinia actin tail-forming complex. Recruitment of Nck and Grb2 to viral particles requires phosphorylation of tyrosine residues 112 and 132 of A36R, the vaccinia actin tail nucleator, respectively. The presence of Grb2 on the virus is also dependent on the polyproline-rich region of N-WASP. The Grb2 pathway alone is therefore unable to nucleate actin tails, as its recruitment requires the prior recruitment of N-WASP by Nck. However, Grb2 does play an important role in actin-based motility of vaccinia, as in its absence, the mean number of actin tails per cell is reduced 2.6-fold. Thus, both Nck and Grb2 act in a cooperative manner to stabilize and/or activate the vaccinia actin-nucleating complex. We suggest that such cooperativity between "primary" and "secondary" adaptor proteins is likely to be a general feature of receptor-mediated signalling.
