Internalization of mammalian fluorescent cellular prion protein and N-terminal deletion mutants in living cells.
The cellular prion protein (PrP(c)) is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein whose conformational altered forms (PrP(sc)) are known to cause neurodegenerative diseases in mammals. In order to investigate the intracellular traffic of mammalian PrP(c) in living cells, we have generated a green fluorescent protein (GFP) tagged version of PrP(c). The recombinant protein was properly anchored at the cell surface and its distribution pattern was similar to that of the endogenous PrP(c), with labeling at the plasma membrane and in an intracellular perinuclear compartment. Comparison of the steady-state distribution of GFP-PrP(c) and two N-terminal deletion mutants (Delta32-121 and Delta32-134), that cause neurological symptoms when expressed in PrP knockout mice, was carried out. The mutant proteins accumulated in the plasma membrane at the expense of decreased labeling in the perinuclear region when compared with GFP-PrP(c). In addition, GFP-PrP(c), but not the two mutants, internalized from the plasma membrane in response to Cu2+ treatment and accumulated at a perinuclear region in SN56 cells. Our data suggest that GFP-PrP(c) can be used to follow constitutive and induced PrP(c) traffic in living cells.
Lee
K S
KS
Ludwig Institute for Cancer Research, São Paulo Branch, Rua Antonio Prudente, São Paulo, Brazil.
Magalhães
A C
AC
Zanata
S M
SM
Brentani
R R
RR
Martins
V R
VR
Prado
M A
MA
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D016296:Mutagenesis; D018621:PrPC Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003300:Copper