Lysosomal membrane transporter purification and reconstitution for functional studies.

Lysosomes achieve their function through numerous transporters that import or export nutrients across their membrane. However, technical challenges in membrane protein overexpression, purification, and reconstitution hinder our understanding of lysosome transporter function. Here, we developed a platform to overexpress and purify the putative lysine transporter Ypq1 using a constitutive overexpression system in protease- and ubiquitination-deficient yeast vacuoles. Using this method, we purified and reconstituted Ypq1 into proteoliposomes and showed lysine transport function, supporting its role as a basic amino acid transporter on the vacuole membrane. We also found that the absence of lysine destabilizes purified Ypq1 and causes it to aggregate, consistent with its propensity to be downregulated in vivo upon lysine starvation. Our approach may be useful for the biochemical characterization of many transporters and membrane proteins to understand organellar transport and regulation.

The In Vitro Cytotoxic Effects of Ionophore Exposure on Selected Cytoskeletal Proteins of C2C12 Myoblasts.

Carboxylic ionophores, such as monensin, salinomycin and lasalocid, are polyether antibiotics used widely in production animals for the control of coccidiosis, as well as for the promotion of growth and feed efficiency. Although the benefits of using ionophores are undisputed, cases of ionophore toxicosis do occur, primarily targeting the cardiac and skeletal muscles of affected animals. The 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) viability assay was used to determine the cytotoxicity of monensin, salinomycin and lasalocid on mouse skeletal myoblasts (C2C12). Immunocytochemistry and immunofluorescent techniques were, in turn, performed to investigate the effects of the ionophores on the microfilament, microtubule and intermediate filament, i.e., desmin and synemin networks of the myoblasts. Monensin was the most cytotoxic of the three ionophores, followed by salinomycin and finally lasalocid. Monensin and salinomycin exposure resulted in the aggregation of desmin around the nuclei of affected myoblasts. The synemin, microtubule and microfilament networks were less affected; however, vesicles throughout the myoblast's cytoplasm produced gaps within the microtubule and, to a limited extent, the synemin and microfilament networks. In conclusion, ionophore exposure disrupted desmin filaments, which could contribute to the myofibrillar degeneration and necrosis seen in the skeletal muscles of animals suffering from ionophore toxicosis.

In Vitro Cytotoxicity Induced by the Bufadienolides 1α,2α-Epoxyscillirosidine and Lanceotoxin B on Rat Myocardial and Mouse Neuroblastoma Cell Lines.

Consumption of bufadienolide-containing plants are responsible for many livestock mortalities annually. Bufadienolides are divided into two groups; non-cumulative bufadienolides and cumulative bufadienolides. Cumulative bufadienolides are referred to as neurotoxic, as the chronic intoxication with this type of bufadienolide results in a paretic/paralytic syndrome known as 'krimpsiekte'. The in vitro cytotoxicity of a non-cumulative bufadienolide, 1α,2α-epoxyscillirosidine, and a cumulative bufadienolide, lanceotoxin B, were compared using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction) assay after exposing rat myocardial (H9c2) and mouse neuroblastoma (Neuro-2a) cell lines. The effect of these two bufadienolides on cell ultrastructure was also investigated using transmission electron microscopy (TEM). H9c2 cells exhibited greater cytotoxicity when exposed to 1α,2α-epoxyscillirosidine, compared to lanceotoxin B. In contrast, Neuro-2a cells were more susceptible to lanceotoxin B. The EC50 (half maximal effective concentration) of lanceotoxin B exposure of Neuro-2a cells for 24⁻72 h ranged from 4.4⁻5.5 µM compared to EC50s of 35.7⁻37.6 µM for 1α,2α-epoxyscillirosidine exposure of Neuro-2a cells over the same period. 1α,2α-Epoxyscillirosidine induced extensive vacuolization in both cell types, with swollen RER (rough endoplasmic reticulum) and perinuclear spaces. Lanceotoxin B caused swelling of the mitochondria and sequestration of cytoplasmic material within autophagic vesicles. These results corroborate the notion that cumulative bufadienolides are neurotoxic.