Abstract
The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs.
Highlights
During the early stages of intracellular protein transport, nascent secretory and integral membrane proteins are synthesized at cytosolic ribosomes and must be targeted to the membrane of the endoplasmic reticulum (ER), a process which is mediated by the signal recognition particle (SRP)-Sec61 targeting/translocation pathway, a well described mechanism even at the structural level [1, 2]
It was experimentally confirmed that it passes the SRP-Sec61 targeting/translocation pathway by using its cleavable signal peptide [27]
Another advantage of taking a G protein coupled receptors (GPCRs) is that these receptors form the largest protein family in eukaryotic cells, which allows later on to analyze cross-selectivity of identified compounds on comparable protein targets
Summary
During the early stages of intracellular protein transport, nascent secretory and integral membrane proteins are synthesized at cytosolic ribosomes and must be targeted to the membrane of the endoplasmic reticulum (ER), a process which is mediated by the SRP-Sec targeting/translocation pathway, a well described mechanism even at the structural level [1, 2]. Following a tight interaction between the ribosome and the Sec complex, the signal sequence is involved in destabilization of the closed conformation of Sec61α causing the relocation of its plug domain (translocon gating). Translation accelerates, secretory proteins are translocated through the protein-conducting channel into the ER lumen whereas their signal peptides leave the protein-conducting channel at a lateral gate. Through this cleavage, secretory proteins are released into the ER lumen. Signal anchor sequences a well as the other transmembrane domains of integral membrane proteins are released through the lateral gate of the protein-conducting channel and become part of the ER membrane. The Sec complex is stably and transiently associated with supporting proteins such as the binding immunoglobulin protein (BiP, HSP70 chaperone family), Sec (HSP40 chaperone family), the translocating chain-associated membrane protein (TRAM), the translocon-associated protein complex (TRAP) and the oligosaccharyl transferase complex (OST)
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