Assuntos
Pseudo-Hipoparatireoidismo/genética , Deleção de Sequência , Sintaxina 16/genética , Adulto , Cromograninas/genética , Éxons/genética , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Heterozigoto , Humanos , Reação em Cadeia da Polimerase Multiplex , Pseudo-Hipoparatireoidismo/complicações , Sintaxina 16/deficiência , Tetania/etiologiaRESUMO
The retrograde transport pathways from early/recycling endosomes are critical for recycling a range of endogenous cargo, as well as internalisation of bacterial and plant toxins. We have previously shown that the retrograde transport of the two model cargos, TGN38 and Shiga toxin, differs in the requirement for TGN golgins; transport of TGN38 requires the TGN golgin GCC88 whereas that of Shiga toxin requires GCC185. Here we have further defined the retrograde transport requirements of these two cargos. Tracking the transport of these cargos demonstrated that the bulk of Shiga toxin is transported from early endosomes to recycling endosomes en route to the TGN whereas the bulk of TGN38 is transported from early endosomes to the TGN with only low levels detected in recycling endosomes. In cells depleted of the TGN t-SNARE syntaxin 16, TGN38 accumulated predominantly in early endosomes whereas Shiga toxin accumulated in Rab11-positive recycling endosomes, suggesting distinct routes for each cargo. Retrograde transport of Shiga toxin and TGN38 requires retromer, however, whereas sorting nexin 1 (SNX1) is specifically required for transport of Shiga toxin, sorting nexin 2 (SNX2) is required for the transport of TGN38. Overall, our data have identified different itineraries for the retrograde transport of Shiga toxin and TGN38 and distinct retromer components that regulate the transport of these cargos.
Assuntos
Endossomos/metabolismo , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Toxina Shiga/metabolismo , Transporte Biológico , Compartimento Celular , Endocitose , Células HeLa , Humanos , Nexinas de Classificação , Sintaxina 16/deficiência , Sintaxina 16/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Syntaxin 10 is a soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein localized to the trans-Golgi network (TGN), where two other members of the syntaxin family, syntaxins 6 and 16, also reside. The role of syntaxin 10 in regulating TGN protein traffic is not yet defined. Syntaxin 10 co-localizes well with syntaxins 6 and 16 at the TGN in interphase cells, and appears to interact with both syntaxin 6 and 16 as evidenced by co-immunoprecipitation analyses. However, unlike syntaxin 6 and 16, neither syntaxin 10 antibodies nor its cytosolic domain inhibits endosome-TGN transport of shiga toxin. Syntaxin 16 knockdown with small interfering RNA (siRNA) affects the TGN localization of syntaxin 6 but not syntaxin 10, and clearly inhibits endosome-TGN transport. On the other hand, knockdown of syntaxin 10 expressions had no significant effect on the TGN localization of syntaxin 6 and 16, and did not inhibit endosome-TGN transport. Unlike syntaxin 16, syntaxin 10 does not interact specifically with Vps45, the Sec1/Munc18 (SM) family member at the TGN. On the other hand, syntaxin 10 reciprocally co-immunoprecipitated endosomal syntaxin 12/13, and knockdown of syntaxin 10 expressions affects the surface expression of transferrin receptor (TfR) and seems to induce the formation of an immobile TfR pool. Therefore, in spite of its co-localization and possible interaction with syntaxin 16, syntaxin 10 is not part of the syntaxin 16-based SNARE complex involved in endosome-TGN transport, and may have a hitherto unrecognized function in the TGN-endosome boundary.