Inhibitors of the Sec61 Complex and Novel High Throughput Screening Strategies to Target the Protein Translocation Pathway

Abstract

Proteins targeted to the secretory pathway start their intracellular journey by being transported across biological membranes such as the endoplasmic reticulum (ER). A central component in this protein translocation process across the ER is the Sec61 translocon complex, which is only intracellularly expressed and does not have any enzymatic activity. In addition, Sec61 translocon complexes are difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its function has thus been notoriously difficult. However, such translocation inhibitors may not only be valuable tools for cell biology, but may also represent novel anticancer drugs, given that cancer cells heavily depend on efficient protein translocation into the ER to support their fast growth. In this review, different inhibitors of protein translocation will be discussed, and their specific mode of action will be compared. In addition, recently published screening strategies for small molecule inhibitors targeting the whole SRP-Sec61 targeting/translocation pathway will be summarized. Of note, slightly modified assays may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex, in order to identify novel antibiotic drugs.

Keywords: Sec61 dependent translocation; co-translational translocation; endoplasmic reticulum; high throughput screening; inhibitor; signal recognition particle dependent protein targeting.

Figure 1

Overview of the secretory pathway for protein biogenesis and topology-based classification of secretory and single pass membrane proteins. Cleavable N-terminal SPs target secretory and type I membrane proteins to the ER membrane, resulting in an N-terminally translocated topology. In the case of type II and type III membrane proteins, the TMD functions as a targeting sequence. Depending on the overall hydrophobicity and charge of the topological sequences adjacent to the TMD, the C- or N-terminal end of the protein is translocated into the ER lumen (type II and type III single pass membrane protein, respectively). Type IV, or TA proteins, are targeted to the ER membrane via the C-terminal TMD. As a result, TA proteins are post-translationally translocated via the TRC-40 pathway. SP: signal peptide, ER: endoplasmic reticulum, TMD: transmembrane domain, TA: tail-anchored, TRC-40: transmembrane recognition complex subunit of 40 kDa.

Figure 2

Overview of the SRP dependent pathway for co-translational translocation via the Sec61 translocon. A secretory or integral membrane protein is targeted toward the ER membrane by means of SRP binding to the signal sequence (i.e., the SP or TMD (steps 1–2)). SRP binding stalls protein translation to keep the nascent chain in a translocation competent state. At the ER membrane, SRP interacts with the SRP receptor. The RNC complex is then transferred to the Sec61 translocon (step 3). Interaction of the ribosome with the translocon reinitiates translation and induces conformational changes within Sec61α, eventually leading to protein translocation. In the case of a weak hydrophobic SP or TMD, the protein requires help from accessory proteins such as TRAP, TRAM, Sec62, and/or Sec63 for protein translocation. In the ER lumen, the SP is cleaved by the signal peptidase complex and the protein is glycosylated by the OST complex (step 4). SRP: signal recognition particle, ER: endoplasmic reticulum, SP: signal peptide, TMD: transmembrane domain, RNC: ribosomal nascent chain, TRAP: translocon-associated protein, TRAM: translocating chain-associating membrane protein, OST: oligosaccharyl transferase.

Figure 3

Dynamics of the TMHs of the Sec61 translocon (PDB 5A6U [69]) upon binding of the ribosome (primed state, PDB 3J7Q [52]) and insertion of the SP (engaged state, PDB 3JC2 [27]). Sec61α is shown in grey, Sec61β is shown in dark grey, and Sec61γ is shown in black. The interhelical interaction between TMH2 and TMH3 (shown in blue) on one half of the translocon, and TMH7 and TMH8 (shown in green) on the other half of the translocon form the lateral gate of Sec61α. Additionally, the translocon is closed axially by the lumenal plug domain of TMH2 (shown in red). Binding of the ribosome disrupts the interaction of TMH3 and TMH8 of the lateral gate and primes the translocon for insertion of the nascent protein chain, while the plug domain remains in place. The SP of the nascent chain (shown in pink) interacts with the lateral gate of the translocon, resulting in lateral escape from the translocon and insertion of the TMD into the ER membrane. In addition, the plug domain is displaced to allow for protein translocation into the ER lumen. Resistance conferring mutations located in the lateral gate or plug domain of Sec61α are shown in ‘Resistant Sec61’. TMH: transmembrane helix, SP: signal peptide, TMD: transmembrane domain, ER: endoplasmic reticulum.

Figure 4

Scheme of the primary and the secondary screen in stably transfected HEK 293 cells. In the primary screen (a), Tet-On-controlled CRF1R.GFP was used, a GFP-tagged GPCR possessing a cleavable signal peptide that uses the SRP-Sec61 targeting/translocation pathway. The secondary screen (b) was performed with Tet-On-controlled, unfused, soluble GFP, which does not use the SRP-Sec61 targeting/translocation pathway. Hits of the primary screen were used in the secondary screen to deselect inhibitors of the transcription/translation machinery. Figure modified from [67].