Introduction In eukaryotic cells, the protein secretory pathway initiates with transport across the endoplasmic reticulum (ER) membrane. Two major pathways have been described. In the first one, the preprotein is not released in the cytoplasm but is directly transferred into the ER lumen in a process called co- translational translocation. The ribosome-nascent chain complex is recognized and targeted to the ER membrane by the signal recognition particle (Walter and Blobel, 1980) and docks at the translocation site. Translation then provides the energy for precursor progression through the ER membrane (Nicchitta and Blobel, 1993). In the second pathway, which has been well studied in the yeast Saccharomyces cerevisiae, proteins are translocated post-translationally in an unfolded state (Rothblatt and Meyer, 1986). In this mode, cytosolic heat shock proteins (HSP) belonging to the HSP70 family interact with the nascent protein to prevent its folding (Deshaies et al., 1988), and the translocation machinery specifically recognises the secretory protein at the ER membrane and actively pulls the precursor into the lumen of the ER (Panzner et al., 1995; Matlack et al., 1999). The translocation sites that allow transport of hydrophilic proteins across a hydrophobic membrane are aqueous channels (Simon and Blobel, 1991), formed by the oligomerization of a trimeric complex, the Sec61 complex (Hanein et al., 1996). The first subunit of this complex, Sec61α, was initially identified in S. cerevisiae as Sec61p (Deshaies and Schekman, 1987); it was later discovered in mammalian cells too and displays strong homology with the Escherichia coli SecY protein (Görlich et al., 1992). The integral Sec61α protein contains several transmembrane domains that were found in proximity to the nascent chains during their transfer and were shown to contribute to the hydrophilic environment reported in translocation pores (Mothes et al., 1994). Sec61β and Sec61γ were co-purified in complex with the Sec61α polypeptide in mammals (Görlich and Rapoport, 1993). In S. cerevisiae, the γ subunit, Sss1p, was isolated as a suppressor of the sec61-2 temperature-sensitive mutation (Esnault et al., 1993). This single transmembrane domain protein is related to the SecE subunit of E. coli translocase (Hartmann et al., 1994). Although both polypeptides are encoded by essential genes in the yeast, the third one, Sbh1p (S ec61β h omolog) (Panzner et al., 1995), does not display an essential function (Finke et al., 1996). A second trimeric complex was identified in S. cerevisiae comprising Ssh1p (a S ec6 1p h omolog) and Sbh2p (another Sec61β homolog) proteins together with Sss1p. In vitro studies indicate that this second Sec61 complex was specialized in the co-translational translocation pathway (Finke et al., 1996). Yeast Sss1p and mammalian Sec61γ proteins are highly conserved and were shown to be functionally interchangeable (Esnault et al., 1993). By contrast, Sbh1p and Sec61β show poor homology and are not related to the third component of E. coli translocation apparatus, SecG. Until now, no precise function has been attributed to the β subunit of the Sec61 complex in either yeast or higher eukaryotic cells. We addressed the function of Sec61 in the yeast Yarrowia 4947 The core component of the translocation apparatus, Sec61p or α, was previously cloned in Yarrowia lipolytica. Using anti-Sec61p antibodies, we showed that most of the translocation sites are devoted to co-translational translocation in this yeast, which is similar to the situation in mammalian cells but in contrast to the situation in Saccharomyces cerevisiae, where post-translational translocation is predominant. In order to characterize further the minimal translocation apparatus in Y. lipolytica, the β Sec61 complex subunit, Sbh1p, was cloned by functional complementation of a ∆sbh1, ∆sbh2 S. cerevisiae mutant. The secretion of the reporter protein is not impaired in the Y. lipolytica sbh1 inactivated strain. We screened the Y. lipolytica two-hybrid library to look for partners of this translocon component. The ER- membrane chaperone protein, calnexin, was identified as an interacting protein. By a co-immunoprecipitation approach, we confirmed this association in Yarrowia and then showed that the S. cerevisiae Sbh2p protein was a functional homologue of YlSbh1p. The interaction of Sbh1p with calnexin was shown to occur between the lumenal domain of both proteins. These results suggest that the β subunit of the Sec61 translocon may relay folding of nascent proteins to their translocation. Key words: Translocation, Quality control, Sec61 β Summary Sbh1p, a subunit of the Sec61 translocon, interacts with the chaperone calnexin in the yeast Yarrowia lipolytica Anita Boisramé*, Marion Chasles, Anna Babour, Jean-Marie Beckerich and Claude Gaillardin Laboratoire de Génétique moléculaire et cellulaire, INRA, CNRS, Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France *Author for correspondence (e-mail: boisrame@grignon.inra.fr) Accepted 24 September 2002 Journal of Cell Science 115, 4947-4956 © 2002 The Company of Biologists Ltd doi:10.1242/jcs.00187 Research Article