Selenoprotein K Is a Peripheral ER Membrane Protein

Selenoprotein K (selenok) is a small, disordered membrane protein associated with the endoplasmic reticulum (ER) that is involved in protein palmitoylation and protein quality control. Through these processes, it influences calcium homeostasis, cellular migration, and phagocytosis. Thus, it is implicated in cancer, neurodegenerative diseases, and autophagy. So far, selenok has been considered a single-pass membrane protein whose N-terminus is in the ER lumen while its C-terminus, which contains the reactive selenocysteine, is in the cytoplasm. Here, we show that selenok is, in fact, a peripheral membrane protein that is anchored to the cytoplasmic side of the ER membrane. We demonstrate, using immunofluorescence microscopy and the substituted cysteine accessibility method in combination with selective membrane permeabilization, that both selenok’s N- and C-terminus are in the cytoplasm. Using the same techniques, we demonstrate that, in contrast, selenoprotein S (selenos), a functionally related member of the selenoprotein family, is a transmembrane protein with a cytoplasmic C-terminus and an N-terminus exposed to the ER lumen. The findings that selenok is a peripheral membrane protein and that its N- and C-terminal segments, along with the hydrophilic side of its amphipathic α-helix, are exposed to the cytoplasm, imply that they can interact with cytoplasmic extramembranous regions of ER-residing membrane proteins and soluble protein partners. Selenok is predicted to possess multiple SLiMs (short linear motifs) involved in protein interactions, and its peripheral topology suggests that all these motifs, including those located within the amphipathic α-helix, are exposed and accessible to cytoplasmic-accessible partners.