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. 2021 Jan 4;267(30):21911–21918. doi: 10.1016/S0021-9258(19)36699-2

Membrane assembly of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation.

JK Locker 1, JK Rose 1, MC Horzinek 1, PJ Rottier 1
PMCID: PMC8740634  PMID: 1400501

Abstract

The M protein of mouse hepatitis virus strain A59 is a triple-spanning membrane protein which assembles with an uncleaved internal signal sequence, adopting an NexoCcyt orientation. To study the insertion mechanism of this protein, domains potentially involved in topogenesis were deleted and the effects analyzed in topogenesis were deleted and the effects analyzed in several ways. Mutant proteins were synthesized in a cell-free translation system in the presence of microsomal membranes, and their integration and topology were determined by alkaline extraction and by protease-protection experiments. By expression in COS-1 and Madin-Darby canine kidney-II cells, the topology of the mutant proteins was also analyzed in vivo. Glycosylation was used as a biochemical marker to assess the disposition of the NH2 terminus. An indirect immunofluorescence assay on semi-intact Madin-Darby canine kidney-II cells using domain-specific antibodies served to identify the cytoplasmically exposed domains. The results show that each membrane-spanning domain acts independently as an insertion and anchor signal and adopts an intrinsic preferred orientation in the lipid bilayer which corresponds to the disposition of the transmembrane domain in the wild-type assembled protein. These observations provide further insight into the mechanism of membrane integration of multispanning proteins. A model for the insertion of the coronavirus M protein is proposed.

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