Monitoring "De Novo"APP synthesis by taking advantage of the reversible effect of cycloheximide.
By blocking "de novo" protein synthesis using cycloheximide, we previously described a dynamic model system to monitor turnover of a specific population of the Alzheimer's amyloid precursor protein. Here we show that cycloheximide is nontoxic and its effect is reversible, allowing protein synthesis to reinitiate. Upon cycloheximide removal protein synthesis restarted and by 1 hour the amyloid precursor protein- green fluorescent protein could be clearly detected, permitting the monitoring of amyloid precursor protein anterograde transport, particularly the secretory pathway. The consensus NPTY motif in amyloid precursor protein, typically associated with endocytosis, was mutated to NPTF or NPTE to mimic a constitutively dephosphorylated or phosphorylated residue, respectively. Our data reveal that disruption of this motif affects amyloid precursor protein endocytosis, as shown previously, but also its incorporation into trans-Golgi network budding vesicles. Thus, cycloheximide can be a useful tool to study both anterograde and retrograde "in vivo'' protein transport.
Rebelo
Sandra
S
Laboratório de Neurociências Centro de Biologia Celular, Universidade de Aveiro, Aveiro, Portugal.
Vieira
Sandra Isabel
SI
da Cruz E Silva
Edgar F
EF
da Cruz E Silva
Odete A B
OA
D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003513:Cycloheximide / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D055571:Secretory Pathway / Q000187:drug effects*; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003513:Cycloheximide