The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment

The heteromeric Sec61 complex is composed of alpha, beta and gamma subunits and forms the core of the mammalian ER translocon, Oligomers of the Sec61 complex form a transmembrane channel where proteins are translocated across and integrated into the ER membrane. We have studied the subcellular local isation of the Sec61 complex using both wild-type COS1 cells and cells tran sfected with GFP-tagged Sec61 alpha, By double labelling immunofluorescence microscopy the GFP-tagged Sec61 alpha was found in both the ER and the ER- Golgi intermediate compartment (ERGIC) but not in the trans-Golgi network. Immunofluorescence studies of endogenous Sec61 beta and Sec61 gamma showed that these proteins are also located in both the ER and the ERGIC, Using th e alternative strategy of subcellular fractionation, we have shown that wil d-type Sec61 alpha, beta and gamma, and GFP-tagged Sec61 alpha, are all pre sent in both the ER and the ERGIC/Golgi fractions of the gradient. The pres ence of the Sec61 subunits in a post-ER compartment suggests that these pro teins can escape the ER and be recycled back, despite the fact that none of them contain any known membrane protein retrieval signals such as cytosoli c di-lysine or di-arginine motifs, We also found that another translocon co mponent, the glycoprotein TRAM, was present in post-ER compartments as demo nstrated by subcellular fractionation, Our data indicate that the core comp onents of the mammalian ER translocon are not permanently resident in the E R, but rather that they are maintained in the ER by a specific retrieval me chanism.