ABSTRACT
The enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is normally localized in the endoplasmic reticulum (ER) where it can esterify cholesterol for storage in lipid droplets and/or the formation of lipoproteins. Here, we report that ACAT can translocate from the ER into vesicular structures in response to different ACAT inhibitors. The translocation was fast (within minutes), reversible and occurred in different cell types. Interestingly, oleic acid was able to fasten the re-translocation from vesicles back into the reticular ER network. The process of ACAT translocation could also be induced by cyclodextrins, cholesterol, lanosterol (but not 4-cholestene-3 one), 25-hydroxycholesterol, and by certain stress stimuli such as hyperosmolarity (sucrose treatment), temperature change, or high-density cultivation. In vitro esterification showed that ACAT remains fully active after it has been translocated to vesicles in response to hyperosmotic sucrose treatment of the cells. The translocation process was not accompanied by changes in the electrophoretic mobility of ACAT, even after chemical crosslinking. Interestingly, the protein synthesis inhibitor cycloheximide showed a stimulating effect on ACAT activity and prevented the translocation of ACAT from the ER into vesicles.
ABSTRACT
Human Claudin-7 (Cldn7) is a member of the Claudin (Cldn) superfamily. In vivo, these proteins form tight junctions, which establish constricted connections between cells. Cldns oligomerize within the membrane plane (= cis-interaction), and also interact with Cldns from adjacent cells (= trans-interaction). Interactions of Cldns are typically studied in vivo and structural analyses of isolated Cldns are limited. Here, we describe heterologous expression in E. coli and purification of human Cldn7, enabling in vitro analyses of the isolated protein using detergent and model membrane systems. Cldn7 exists as a monomer, hexamer, and various higher oligomers in micelles. While only limited unfolding of the protein was observed in the presence of the anionic detergent sodium dodecyl sulfate, decreased ionic strength did affect Cldn7 cis-interactions. Furthermore, we identified two amino acids which mediate electrostatic cis-interactions and analyzed the impact of disturbed cis-interaction on trans-contacts via atomic force microscopy and monitoring Förster resonance energy transfer between fluorescently labeled Cldn7-containing proteoliposomes. Our results indicate that Cldn7 cis-oligomerization might not be a prerequisite for establishing trans-contacts.
ABSTRACT
The spatial distribution of proteins in cell membranes is crucial for signal transduction, cell communication and membrane trafficking. Members of the Tetraspanin family organize functional protein clusters within the plasma membrane into so-called Tetraspanin-enriched microdomains (TEMs). Direct interactions between Tetraspanins are believed to be important for this organization. However, studies thus far have utilized mainly co-immunoprecipitation methods that cannot distinguish between direct and indirect, through common partners, interactions. Here we study Tetraspanin 8 homointeractions in living cells via quantitative fluorescence microscopy. We demonstrate that Tetraspanin 8 exists in a monomer-dimer equilibrium in the plasma membrane. Tetraspanin 8 dimerization is described by a high dissociation constant (Kd = 14 700 ± 1100â Tspan8/µm2), one of the highest dissociation constants measured for membrane proteins in live cells. We propose that this high dissociation constant, and thus the short lifetime of the Tetraspanin 8 dimer, is critical for Tetraspanin 8 functioning as a master regulator of cell signaling.
