Subcellular imaging of MGAT1/MGAT2 homo- and heteromers in living cells using bioluminescence microscopy.
Biochem Biophys Res Commun
; 734: 150470, 2024 Jul 29.
Article
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| MEDLINE
| ID: mdl-39083973
ABSTRACT
Protein-protein interactions (PPIs) play fundamental roles in many biological processes including the functioning of glycosylation machineries present in the endoplasmic reticulum (ER) and Golgi apparatus of mammalian cells. For the last couple of years, we have been successfully employing the most advanced version of the split luciferase complementation assay, termed NanoBiT, to demonstrate PPIs between solute carrier 35 (SLC35) family members with nucleotide sugar transporting activity and functionally related glycosyltransferases. NanoBiT has several unmatched advantages as compared with other strategies for studying PPIs. Firstly, the tendency of the free luciferase fragments to spontaneously associate is strongly reduced. As a consequence, the fragments of the reconstituted luciferase may dissociate upon the disruption of the PPI of interest. Secondly, the recombinant fusion proteins are expressed at low (near-endogenous) levels. Both of these features significantly minimize the possibility of obtaining false positive results. In this study we pushed the boundaries of this already powerful technique even further by coupling it with bioluminescence imaging of PPIs. Specifically, we visualized homo- and heterologous complexes formed by MGAT1 and MGAT2 glycosylation enzymes tagged with NanoBiT fragments and demonstrated ER-to-Golgi transitions between enzyme homo- and heteromers.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
Biochem Biophys Res Commun
Año:
2024
Tipo del documento:
Article
País de afiliación:
Polonia
Pais de publicación:
Estados Unidos