ABSTRACT
The endoplasmic reticulum (ER) is the entry point to the secretory pathway and major site of protein biogenesis. Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the evolutionarily conserved Sec61 complex, a heterotrimeric channel that comprises the Sec61p/Sec61α, Sss1p/Sec61γ, and Sbh1p/Sec61ß subunits. In addition to forming a protein-conducting channel, the Sec61 complex also functions to maintain the ER permeability barrier, preventing the mass free flow of essential ER-enriched molecules and ions. Loss in 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 we hypothesize it is important for gating the ER translocon. The ER stress response was found to be constitutively induced in two temperature-sensitive sss1 mutants (sss1ts ) that are still proficient to conduct ER translocation. A screen to identify intergenic mutations that allow for sss1ts cells to grow at 37 °C suggests the ER permeability barrier to be compromised in these mutants. We propose the extreme C terminus of Sss1p/Sec61γ is an essential component of the gating module of the ER translocase and is required to maintain the ER permeability barrier.
Subject(s)
Endoplasmic Reticulum/genetics , Protein Biosynthesis/genetics , SEC Translocation Channels/genetics , Saccharomyces cerevisiae Proteins/genetics , Amino Acid Sequence/genetics , Endoplasmic Reticulum Stress/genetics , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Mutation/genetics , Permeability , Protein Transport/genetics , SEC Translocation Channels/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
BiP (Kar2 in yeast) is an essential Hsp70 chaperone and master regulator of endoplasmic reticulum (ER) function. BiP's activity is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, Sil1 and Lhs1. Both Sil1 and Lhs1 are glycoproteins, but how N-glycosylation regulates their function is not known. Here, we show that N-glycosylation of Sil1, but not of Lhs1, is diminished upon reductive stress. N-glycosylation of Sil1 is predominantly Ost3-dependent and requires a functional Ost3 CxxC thioredoxin motif. N-glycosylation of Lhs1 is largely Ost3-independent and independent of the CxxC motif. Unglycosylated Sil1 is not only functional but is more effective at rescuing loss of Lhs1 activity than N-glycosylated Sil1. Furthermore, substitution of the redox active cysteine pair C52 and C57 in the N terminus of Sil1 results in the Doa10-dependent ERAD of this mutant protein. We propose that reductive stress in the ER inhibits the Ost3-dependent N-glycosylation of Sil1, which regulates specific BiP functions appropriate to the needs of the ER under reductive stress.