Members of a mammalian SNARE complex interact in the endoplasmic reticulum in vivo and are found in COPI vesicles.
Retrograde traffic between the Golgi apparatus and the endoplasmic reticulum (ER) is largely mediated by COPI-coated transport vesicles. In mammalian cells, retrograde traffic can pass through an intermediate compartment. Here, we report that the mammalian soluble N-ethylmaleimide-sensitive factor (NSF) attachment receptor (SNARE) proteins mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 form a quaternary SNARE complex. Fluorescence resonance energy transfer (FRET) experiments prove that these interactions occur in the ER of living cells. In addition, mUse1/D12 and mSec20/BNIP1 form homo-oligomers in vivo. Furthermore, we show that mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 are recruited into COPI-coated vesicles formed in vitro. Immunogold electron microscopy confirmed that these SNARE proteins colocalize with the KDEL receptor ERD2 in COPI-coated vesicles. Moreover, both FRET and immunoprecipitation experiments reveal interactions of these SNAREs with both ERD2 and COPI subunits. We conclude that the SNAREs described here are sorted via interaction with components of the COPI-dependent budding complex into Golgi-to-ER retrograde COPI vesicles and function in retrograde transport from the Golgi to the ER Golgi intermediate compartment (ERGIC) or the ER.
Department of Neurobiology, Max-Planck-Institute of Biophysical Chemistry, Göttingen.
Sophie Estelle
SE
Verrier
Matthias
M
Willmann
Dirk
D
Wenzel
Ulrike
U
Winter
Gabriele Fischer
GF
von Mollard
Hans-Dieter
HD
Söling
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D022181:COP-Coated Vesicles / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D050767:Qc-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D033921:Vesicular Transport Proteins
C485597:Bnip1 protein, rat; D019253:Proto-Oncogene Proteins c-bcl-2; D050765:Qa-SNARE Proteins; D050767:Qc-SNARE Proteins; D050600:SNARE Proteins; C539363:Use1 protein, mouse; D033921:Vesicular Transport Proteins