Caveolin-1β promotes the production of active human microsomal glutathione S-transferase in induced intracellular vesicles in Spodoptera frugiperda  insect cells

Perrot, Nahuel; Dessaux, Delphine; Rignani, Anthony; Gillet, Cynthia; Orlowski, Stéphane; Jamin, Nadège; Garrigos, Manuel; Jaxel, Christine

doi:10.1016/j.bbamem.2022.183922

Cited by 4 publications

(6 citation statements)

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“…The gene of canine caveolin-1β (Cav1β, Uniprot P33724.2, 32–178) cloned into the pKL vector [ 20 ] with a 10-His affinity tag and TEV protease site at the N-terminus of the gene (HTC for His-tag-TEV-caveolin) was used for subcloning into pNZ8148. First, a mutation was performed to insert the restriction site Nco I at the start codon using the QuickChange Lightning Site-Directed Mutagenesis kit (Agilent) following the manufacturer’s instructions with the primer pKL-NcoIm fwd and rev (Table 2 ).…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, it is also essential to provide fundamental insights into the formation of specific domains on membrane, the deformation of a biological membrane driven by caveolin-1, the organization of a caveolar coat, and the requirement of specific lipids and proteins during the process. Caveolin-1, isoforms αand β have been expressed in E. coli [ 16 , 17 ] , insect cells [ 18 – 20 ], Toxoplasma gondii [ 21 ], and cell-free system [ 22 ]. In all these systems (except cell-free), the expression of the recombinant caveolin induced the formation of intracellular vesicles within the heterologous host.…”

Section: Introductionmentioning

confidence: 99%

“…Notably, in these few expression systems, both whole and truncated versions of caveolin-1 have been demonstrated to be sufficient alone to promote membrane budding and protrusion, and the eventual formation of intracellular vesicles presenting high homogeneity of size and shape, provided local concentration of the protein is high enough [ 23 ]. Such intracellular vesicles, called “heterologous caveolae” [ 17 ], could be used for diverse biotechnological purposes, and especially for co-expression of other membrane proteins (MPs) of interest [ 20 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

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Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

et al. 2022

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Background Caveolae are invaginated plasma membrane domains of 50–100 nm in diameter involved in many important physiological functions in eukaryotic cells. They are composed of different proteins, including the membrane-embedded caveolins and the peripheric cavins. Caveolin-1 has already been expressed in various expression systems (E. coli, insect cells, Toxoplasma gondii, cell-free system), generating intracellular caveolin-enriched vesicles in E. coli, insect cells and T. gondii. These systems helped to understand the protein insertion within the membrane and its oligomerization. There is still need for fundamental insights into the formation of specific domains on membrane, the deformation of a biological membrane driven by caveolin-1, the organization of a caveolar coat, and the requirement of specific lipids and proteins during the process. The aim of this study was to test whether the heterologously expressed caveolin-1β was able to induce the formation of intracellular vesicles within a Gram+ bacterium, Lactococcus lactis, since it displays a specific lipid composition different from E. coli and appears to emerge as a good alternative to E. coli for efficient overexpression of various membrane proteins. Results Recombinant bacteria transformed with the plasmid pNZ-HTC coding for the canine isoform of caveolin-1β were shown to produce caveolin-1β, in its functional oligomeric form, at a high expression level unexpected for an eukaryotic membrane protein. Electron microscopy revealed several intracellular vesicles from 30 to 60 nm, a size comparable to E. coli h-caveolae, beneath the plasma membrane of the overexpressing bacteria, showing that caveolin-1β is sufficient to induce membrane vesiculation. Immunolabelling studies showed antibodies on such neo-formed intracellular vesicles, but none on plasma membrane. Density gradient fractionation allowed the correlation between detection of oligomers on Western blot and appearance of vesicles measurable by DLS, showing the requirement of caveolin-1β oligomerization for vesicle formation. Conclusions Lactococcus lactis cells can heterologously overexpress caveolin-1β, generating caveolin-1β enriched intracellular neo-formed vesicles. These vesicles might be useful for potential co-expression of membrane proteins of pharmaceutical interest for their simplified functional characterization.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

et al. 2022

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“…Antibiotics were used for plasmid maintenance at the following nal concentrations: chloramphenicol (10 µg/mL) for L. lactis and kanamycine (100 µg/mL) for E. coli. The gene of canine caveolin-1β (Cav1β, Uniprot P33724.2, 32-178) cloned into the pKL vector [20] with a 10-His a nity tag and TEV protease site at the N-terminus of the gene (HTC for His-tag-TEV-caveolin)…”

Section: Bacterial Strains and Growth Conditionsmentioning

confidence: 99%

“…Notably, in these few expression systems, both whole and truncated versions of caveolin-1 have been demonstrated to be su cient alone to promote membrane budding and protrusion, and the eventual formation of intracellular vesicles presenting high homogeneity of size and shape, if local concentration of the protein is enough [23]. Such intracellular vesicles, called "heterologous caveolae" [17], could be used for diverse biotechnological purposes, and especially for co-expression of other membrane proteins (MPs) of interest [20,24].…”

Section: Introductionmentioning

confidence: 99%

Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

Flourieusse

Bourgeois

Schenckbecker

et al. 2022

Preprint

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Background: Caveolae are invaginated plasma membrane domains of 50-100 nm in diameter involved in many important physiological functions in cells. They are composed of different proteins, including the membrane-embedded caveolins and the peripheric cavins. Caveolin-1 has already been expressed in various expression systems (E. coli, insect cells, Toxoplasma gondii, cell-free system), generating intracellular caveolin-enriched vesicles in E. coli, insect cells and T. gondii. These systems helped to understand the protein insertion within the membrane and its oligomerization. There is still need for fundamental insights into the formation of specific domains on membrane, deformation of a biological membrane driven by caveolin-1, the organization of a caveolar coat, and the requirement of specific lipids and proteins during the process. The aim of this study was to test whether the heterologously expressed caveolin-1β was able to induce the formation of intracellular vesicles within a Gram+ bacterium, Lactococcus lactis, since it displays a specific lipid composition different from E. coli and appears to emerge as a good alternative to E. coli for efficient overexpression of various membrane proteins.Results: Recombinant bacteria transformed with the plasmid pNZ-HTC coding for the canine isoform of caveolin-1β has been shown to produce caveolin-1β, under its functional oligomeric form, at a high expression level unexpected for an eukaryotic membrane protein. Electron microscopy revealed several intracellular vesicles from 30 to 60 nm, a size comparable to E. coli h-caveolae, beneath the plasma membrane of the overexpressing bacteria, showing that caveolin-1β is sufficient to induce membrane vesiculation. Immunolabelling studies showed antibodies on such neo-formed intracellular vesicles, but none on plasma membrane. Density gradient fractionation allowed the correlation between detection of oligomers on Western blotting and appearance of vesicles measurable by DLS, showing the requirement of caveolin-1β oligomerization for vesicle formation. Conclusion: These caveolin-1β enriched intracellular neo-formed vesicles might be useful for potential co-expression of membrane proteins of pharmaceutical interest for their simplified functional characterization.

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The building blocks of caveolae revealed: caveolins finally take center stage

Kenworthy

2023

Biochemical Society Transactions

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The ability of cells to divide, migrate, relay signals, sense mechanical stimuli, and respond to stress all rely on nanoscale invaginations of the plasma membrane known as caveolae. The caveolins, a family of monotopic membrane proteins, form the inner layer of the caveolar coat. Caveolins have long been implicated in the generation of membrane curvature, in addition to serving as scaffolds for signaling proteins. Until recently, however, the molecular architecture of caveolins was unknown, making it impossible to understand how they operate at a mechanistic level. Over the past year, two independent lines of evidence — experimental and computational — have now converged to provide the first-ever glimpse into the structure of the oligomeric caveolin complexes that function as the building blocks of caveolae. Here, we summarize how these discoveries are transforming our understanding of this long-enigmatic protein family and their role in caveolae assembly and function. We present new models inspired by the structure for how caveolins oligomerize, remodel membranes, interact with their binding partners, and reorganize when mutated. Finally, we discuss emerging insights into structural differences among caveolin family members that enable them to support the proper functions of diverse tissues and organisms.

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Caveolin-1β promotes the production of active human microsomal glutathione S-transferase in induced intracellular vesicles in Spodoptera frugiperda insect cells

Cited by 4 publications

References 43 publications

Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

The building blocks of caveolae revealed: caveolins finally take center stage

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