Electron microscopic analysis of a fusion protein of postsynaptic density-95 and metallothionein in cultured hippocampal neurons.
The subcellular localization of biomolecules at high resolution has traditionally been investigated by combining transmission electron microscopy (TEM) and chemical staining with heavy metals or immuno-based labeling with gold-conjugated antibodies. Here, we employ genetically encoded tags to examine the localization of proteins in transfected cultured cells by TEM. We purified a fusion protein of postsynaptic density-95 (PSD-95) coupled to three tandem repeats of metallothionein (MT) (PDS-95-3MT) from COS7 cells grown in the presence of Cd2+. PSD-95-3MT was detected as black particles by TEM. To visualize the subcellular localization of PSD-95-3MT, expression constructs encoding this fusion protein were transfected into primary hippocampal neurons cultured in medium containing Cd2+. The subcellular accumulation of PSD-95-3MT and Cd2+ provided excellent contrast in TEM micrographs. To address if genetically encoded tags affect the function of the target proteins, we found that the conjugation of 3MT to PSD-95 did not alter its association with known binding partners. These results demonstrate that 3MT coordinating Cd2+ is a valuable genetically encoded tag to study the localization of proteins by TEM.
Fukunaga
Yuko
Y
Bio-multisome Research Team, RIKEN SPring8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Hirase
Ai
A
Kim
Hyeji
H
Wada
Natsuko
N
Nishino
Yuri
Y
Miyazawa
Atsuo
A
D000818:Animals; D019556:COS Cells; D002104:Cadmium / Q000737:chemistry / Q000633:toxicity; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000076122:Disks Large Homolog 4 Protein; D051528:Guanylate Kinases; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D051379:Mice; D046529:Microscopy, Electron, Transmission; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D020080:Tandem Repeat Sequences; D014162:Transfection
D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D008668:Metallothionein; D051528:Guanylate Kinases