Abstract
Objective : GATE-16, originally identified as a protein necessary for intra-Golgi transport, has also been shown to be involved in starvation-induced autophagy. In order to better understand the dual functions of GATE-16, we performed a systematic analysis of GATE-16-interacting proteins, using ESI-QTOF (Q-STAR) massspectrometry.
Methods : FLAG-GATE-16 was overexpressed in HEK293 cells and immunoprecipitated with anti-FLAG-M2-agarose. The resultant precipitates were analyzed by SDS-PAGE and identified by silverstaining. Each polypeptide was analyzed using ESI-QTOF (Q-STAR) massspectrometry.
Results : Two proteins (p120, p105) were specifically coimmunoprecipitated with FLAG-GATE-16. The two polypeptides were identified as importin β and GRP94, respectively. When either of the two polypeptides and FLAG-GATE-16 were coexpressed in HEK293 cells, FLAG-GATE 16 was coimmunoprecipitated with importin β or GRP94.
Conclusions : Importin β plays an essential role in protein import from the cytoplasm to the nucleus, whereas GRP94 functions as an endoplasmic reticulum (ER) chaperon. The specific interaction between the two proteins and GATE-16 may indicate that GATE-16 plays a regulatory role in importin β-dependent nuclear import and the uptake of de novo synthesized GRP precursor into ER.
