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1.
J Biol Chem ; 288(40): 28567-80, 2013 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-23965993

RESUMO

The amyloid precursor protein (APP) is a ubiquitously expressed single-pass transmembrane protein that undergoes proteolytic processing by secretases to generate the pathogenic amyloid-ß peptide, the major component in Alzheimer plaques. The traffic of APP through the cell determines its exposure to secretases and consequently the cleavages that generate the pathogenic or nonpathogenic peptide fragments. Despite the likely importance of APP traffic to Alzheimer disease, we still lack clear models for the routing and regulation of APP in cells. Like the traffic of most transmembrane proteins, the binding of adaptors to its cytoplasmic tail, which is 47 residues long and contains at least four distinct sorting motifs, regulates that of APP. We tested each of these for effects on the traffic of APP from the Golgi by mutating key residues within them and examining adaptor recruitment at the Golgi and traffic to post-Golgi site(s). We demonstrate strict specificity for recruitment of the Mint3 adaptor by APP at the Golgi, a critical role for Tyr-682 (within the YENPTY motif) in Mint3 recruitment and export of APP from the Golgi, and we identify LAMP1(+) structures as the proximal destination of APP after leaving the Golgi. Together, these data provide a detailed view of the first sorting step in its route to the cell surface and processing by secretases and further highlight the critical role played by Mint3.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Brefeldina A/farmacologia , Compartimento Celular/efeitos dos fármacos , Furina/metabolismo , Complexo de Golgi/efeitos dos fármacos , Células HeLa , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Mutação/genética , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Temperatura
2.
J Biol Chem ; 288(21): 14788-804, 2013 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-23572528

RESUMO

Membrane traffic requires the specific concentration of protein cargos and exclusion of other proteins into nascent carriers. Critical components of this selectivity are the protein adaptors that bind to short, linear motifs in the cytoplasmic tails of transmembrane protein cargos and sequester them into nascent carriers. The recruitment of the adaptors is mediated by activated Arf GTPases, and the Arf-adaptor complexes mark sites of carrier formation. However, the nature of the signal(s) that initiates carrier biogenesis remains unknown. We examined the specificity and initial sites of recruitment of Arf-dependent adaptors (AP-1 and GGAs) in response to the Golgi or endosomal localization of specific cargo proteins (furin, mannose-6-phosphate receptor (M6PR), and M6PR lacking a C-terminal domain M6PRΔC). We find that cargo promotes the recruitment of specific adaptors, suggesting that it is part of an upstream signaling event. Cargos do not promote adaptor recruitment to all compartments in which they reside, and thus additional factors regulate the cargo's ability to promote Arf activation and adaptor recruitment. We document that within a given compartment different cargos recruit different adaptors, suggesting that there is little or no free, activated Arf at the membrane and that Arf activation is spatially and temporally coupled to the cargo and the adaptor. Using temperature block, brefeldin A, and recovery from each, we found that the cytoplasmic tail of M6PR causes the recruitment of AP-1 and GGAs to recycling endosomes and not at the Golgi, as predicted by steady state staining profiles. These results are discussed with respect to the generation of novel models for cargo-dependent regulation of membrane traffic.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Complexo 1 de Proteínas Adaptadoras/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Receptor IGF Tipo 2/metabolismo , Fatores de Ribosilação do ADP/genética , Complexo 1 de Proteínas Adaptadoras/genética , Animais , Antibacterianos/farmacologia , Brefeldina A/farmacologia , Citoplasma/genética , Citoplasma/metabolismo , Endossomos/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Complexo de Golgi/genética , Células HeLa , Humanos , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Ratos , Receptor IGF Tipo 2/genética
3.
Cell Logist ; 2(4): 176-188, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23538475

RESUMO

The use of fluorescence microscopy is central to cell biology in general, and essential to many fields (e.g., membrane traffic) that rely upon it to identify cellular locations of molecules under study and the extent to which they co-localize with others. Rigorous localization or co-localization data require quantitative image analyses that can vary widely between fields and laboratories. While most published data use two-dimensional images, there is an increasing appreciation for the advantages of collecting three-dimensional data sets. These include the ability to evaluate the entire cell and avoidance of focal plane bias. This is particularly important when imaging and quantifying changes in organelles with irregular borders and which vary in appearance between cells in a population, e.g., the Golgi. We describe a method developed for quantifying changes in signal intensity of one protein within any three-dimensional structure, defined by the presence of a different marker. We use as examples of this method the quantification of adaptor recruitment to transmembrane protein cargos at the Golgi though it can be directly applied to any site in the cell. Together, these advantages facilitate rigorous statistical testing of differences between conditions, despite variations in organelle structure, and we believe that this method of quantification of fluorescence data can be productively applied to a wide array of experimental questions.

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