Rough microsomes isolated from snap-frozen canine pancreatic tissue retain their co-translational translocation functionality.

Abstract

Proteins are essential for life in all organisms: they mediate cell signaling and cell division and provide structure/motility to cells and tissues. All proteins are synthesized on cytoplasmic ribosomes as unfolded precursors that need to find their correct location in the compartmentalized cell. In eukaryotes, ∼30% of the proteome is translocated across or integrated into the endoplasmic reticulum (ER) membrane, a process mostly mediated by the heterotrimeric Sec61 complex that spans the ER membrane. There is significant interest in identifying small-molecule inhibitors of the Sec61 translocon channel that hold great promise as putative anticancer, immunosuppressive, or antiviral drugs. Hence, representative models are needed to study Sec61-dependent protein import into the ER. Microsomal membranes (or microsomes) isolated from dog pancreatic tissue are the primary source of mammalian ER for cell-free in vitro protein translocation research. Here, we demonstrate that for the isolation of microsomal membranes, snap-frozen canine pancreatic tissue can serve as a valuable alternative to freshly isolated organ tissue from euthanized animals. For 17 out of 20 animals, a sufficient yield of microsomes was extracted from defrosted pancreatic tissue. The isolated microsomes contained the essential proteins of the translocation machinery, and proved to be intact as verified by the detection of ER lumenal chaperones. Importantly, 13 out of the 17 microsome samples retained their translocation competence, as reflected by successful in vitro co-translational translocation of wild-type bovine preprolactin. The microsomes supported post-translational modifications of the tested substrates such as signal peptide cleavage and N-linked glycosylation. Furthermore, the tested microsome samples responded well to the translocation inhibitor cyclotriazadisulfonamide in suppressing human CD4 protein translocation into the ER. In conclusion, microsomes isolated from frozen canine pancreatic tissue proved to retain their co-translational translocation functionality that can contribute to our research of Sec61-dependent protein translocation and selective inhibition thereof.