Detecting the surface localization and cytoplasmic cleavage of membrane-bound proteins.
Polycystin-1 (PC1) is a large, membrane-bound protein that localizes to the cilia and is implicated in the common ciliopathy autosomal-dominant polycystic kidney disease. The physiological function of PC1 is dependent upon its subcellular localization as well as specific cleavages that release soluble fragments of its C-terminal tail. The techniques described here allow visualization and quantification of these aspects of the biology of the PC1 protein. To visualize PC1 at the plasma membrane, a live-cell surface labeling immunofluorescence protocol paired with the labeling of an internal antigen motif allows a robust detection of the surface population of this protein. This technique is modified to generate a surface enzyme-linked immunosorbent assay (ELISA), which quantitatively measures the amount of surface protein as a fraction of the total amount of the protein expressed in that cell population. These assays are powerful tools in the assessment of the small but biologically important pool of PC1 that reaches the cell surface. The C-terminal tail cleavage of PC1 constitutes an interesting modification that allows PC1 to extend its functional role into the nucleus. A reporter assay based on Gal4/VP16 luciferase can be used to quantitate the amount of PC1 C-terminal tail that reaches the nucleus. This assay can be paired with quantitative measurement of the protein expression in the cell, allowing a more complete understanding of the pattern of PC1 cleavage and the nuclear localization of the resultant.
Chapin
Hannah C
HC
Department of Cell Biology, Yale University, New Haven, Connecticut 06520, USA.
Rajendran
Vanathy
V
Capasso
Anna
A
Caplan
Michael J
MJ
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000295:instrumentation / Q000379:methods; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050396:TRPP Cation Channels / Q000737:chemistry / Q000378:metabolism*
D008156:Luciferases; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D050396:TRPP Cation Channels