Abstract
Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the Sec61 complex, a heterotrimeric protein complex possessing two essential sub-units, Sec61p/Sec61α and Sss1p/Sec61γ and the non-essential Sbh1p/Sec61β subunit. In addition to forming a protein conducting channel, the Sec61 complex maintains the ER permeability barrier, preventing flow of molecules and ions. Loss of Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C-terminus is juxtaposed to the key gating module of Sec61p/Sec61α and is important for gating the translocon. Inspection of the cancer genome database identifies six mutations in highly conserved amino acids of Sec61γ/Sss1p. We identify that five out of the six mutations identified affect gating of the ER translocon, albeit with varying strength. Together, we find that mutations in Sec61γ that arise in malignant cells result in altered translocon gating dynamics, this offers the potential for the translocon to represent a target in co-therapy for cancer treatment.
Highlights
The endoplasmic reticulum (ER) is the entry point into the secretory pathway [1,2]
Secretory proteins enter the ER via a gated channel in the ER membrane called the translocon, a protein complex composed of Sec61p/Sec61α, Sbh1p/Sec61β and Sss1p/Sec61γ
We have discovered that mutations in Sec61γ that arise in cancer affect this seal but not the ability of this protein complex to translocate secretory proteins into the ER
Summary
Yeast strains (S1 Table) were grown in YP medium (2% peptone, 1% yeast extract) in the presence of 2% glucose (YPD). Growth was predominately performed at 30 ̊C except where defined otherwise for the purposes of TS growth analysis which involved spotting onto media at a 10-fold dilution series. Minimal medium (0.67% yeast nitrogen base; YNB) with the addition of 2% glucose and appropriate supplements (20 μg/ml) was utilised for nutrient selection. 2% (w/v) agar was added for solid media. Minimal media was prepared yet with the addition of 1 g/L 5-fluoroorotic acid (5-FOA) and 100 μg/ml uracil to achieve counter selection of URA3 plasmids. 1 μg/ml terbinafine or DMSO was added to YPD agar where indicated. Site directed mutagenesis was performed according to Q5 Site Directed Mutagenesis Protocol (NEB), the plasmids and oligonucleotides used are listed in S2 and S3 Tables respectively. The plasmid pJKB2 was used as template to introduce the desired mutations into SSS1
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