Expression, localization, and topology of fluorescently tagged plasma membrane-targeted transmembrane proteins.
Transmembrane proteins constitute a significant proportion of the total number of proteins encoded in eukaryotic genomes. These proteins are involved in countless processes required for cellular function and homeostasis. Mutations mapping within their genes form the cellular mechanisms for a variety of pathological conditions. The surface expression of polytopic proteins can be limited by their relatively slow folding in the endoplasmic reticulum (ER). This is especially evident in heterologous overexpression systems . On the other hand, expression of these proteins in endogenously expressing primary cell lines is generally a challenging task in itself. Here we present a comprehensive scheme to establish arrival at the plasma membrane (PM) of transiently expressed, fluorescently tagged transmembrane proteins and provide two experimental tools to determine whether a distinct domain is cytosolic or extracellular using fluorescence microscopy.
Shepshelovich
Jeanne
J
Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel.
Hirschberg
Koret
K
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D018274:Electroporation; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological
D008565:Membrane Proteins; D049452:Green Fluorescent Proteins