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Early steps in protein secretion


In all living organisms, the translocation of the polypeptides through a membrane is a basic process of the secretory pathway of proteins. Manipulation of this process could be economically very important because it appears that many proteins are poorly secreted by heterologous hosts; the understanding of the critical components of the secretory apparatus may enable the construction of specifically engineered host strains in order to maximize the secretion of foreign proteins.

The transport process may be divided in three main steps: first, recruitment and presentation of the polypeptides to the translocation apparatus, secondly translocation through the membrane itself and thirdly refolding of the polypeptide in the endoplasmic reticulum (ER) lumen. Our project has covered these aspects. It has combined results derived from various organisms using very different techniques so that a link will be established between the in vitro tools developed in higher eukaryotes and the genetic tractability of yeasts.
In Y. lipolytica (Lab A), components of the translocation apparatus were identified either by reverse genetics (Sec61, Sec62) or by genetic screening (Sls1p and Tsr1p). Sls1p was identified as a mutant colethal with a thermosensitive form of SRP; it is a component of the ER lumen, but it is tightly involved in the translocation process. Tsr1p, initially characterized as a suppressor mutation of the thermosensitive SRP form is a component of the ER membrane and was shown to interact with SRP and ribosome. An homologue in S. cerevisiae, yhc8p, was shown to be involved in translocation.
Sss1p (LabB), a component of the translocation pore, was shown to span the ER membrane once exposing its amino-terminal half to the cytoplasm. By NMR, this N-terminal domain in canine Sec61gamma (Sss1p homologue) appeared to acquire an alphahelix-loop-alphahelix secondary structure. Sec61p and two other proteins were identified by cross-linking to be in close proximity to Sss1p. Sss1p and Sec61p showed mutually stabilizing interactions. The interactions of these two proteins were mapped to the region covering Sec61p transmembrane segment 8 (in cooperation with Lab C). Srp40p, a multicopy suppressor of a sec61-mutant, localizes to punctate nuclear structures. SRP40 appears to play a dose-sensitive role in preribosome assembly or transport.
In labC, the topology of Sec61p, the main component of the translocation pore was established by a combination of approaches (cloning of homologues in S.pombe and Y.lipolytica (LabA), fusion with a C-terminal reporter protein Suc2p and insertions of factor Xa cleavage sites). A collection of Sec61 mutations was constituted. A novel member of the Hsp70 family Lhs1p localised within the ER lumen was identified. This protein is required for the efficient translocation of a number of secretory protein precursors across the ER membrane.
In LabD, the heptameric and trimeric translocation complexes were purified. A second trimeric complex (Ssh1p complex) was identifiedin yeast. The genes encoding new components Ssh1p, Sbh1p and Sbh2p were cloned and studied. These components have homologues in translocation systems in bacteria, archea and eukaria. The role of these different complexes was established in vivo. An in vitro posttranslational protein trasnslocation system was reconstituted using purified Sec-complex and Kar2p as the only components.

Major scientific breakthroughs
The components of the translocation pores were purified. The structures of some of their components (Sec61p, Sss1p) were described at the molecular level through biochemical experiments and NMR studies. These studies change our view of the translocation process: three translocation pores, two Hsp70 chaperones (Kar2p, Lhs1p) on the lumenal side of the ER, a new polypeptide involved in the docking of SRP displaying a large lumenal domain interacting with Kar2p... The challenge is now to give an integrated view of the translocation machinery and to understand how the different secretory polypeptides are channeled in the two translocation pores.

Call for proposal

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Institut National de la Recherche Agronomique (INRA)
Route De Thiverval
78850 Thiverval-grignon

Participants (3)

Commissariat à l'Energie Atomique
Centre D'etudes De Saclay
91191 Gif-sur-yvette
Robert-roessle-strasse 10
13092 Berlin
University of Manchester
United Kingdom
Oxford Road
M13 9PT Manchester