Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway.
BACKGROUND
MPG is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. In this work, we examined the efficacy of MPG as an N-terminal tag in a fusion protein to deliver a protein cargo and its mechanism of transduction.


RESULTS
We examined transduction of MPG-EGFP fusion protein by live imaging, flow cytometry, along with combination of cell biological and pharmacological methods. We show that MPG-EGFP fusion proteins efficiently enter various mammalian cells within a few minutes and are co-localized with FM4-64, a general marker of endosomes. The transduction of MPG-EGFP occurs rapidly and is inhibited at a low temperature. The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction. Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP. The partial blockade of MPG-EGFP transduction by a dynamin mutant is abolished by the treatment of amiloride. MPG-EGFP transduction is also observed in the mammalian oocytes.


CONCLUSIONS
The results show that the transduction of MPG fusion protein utilizes endocytic pathway(s) which is amiloride-sensitive and partially dynamin-dependent. Collectively, the MPG fusion protein could be further developed as a novel tool of "protein therapeutics", with potentials to be used in various cell systems including mammalian oocytes.
Kwon
So-Jung
SJ
Department of Biomedical Science & Technology IBST Konkuk University 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Korea. ksjpo47@hanmail.net
Han
Kyuyong
K
Jung
Suhyun
S
Lee
Jong-Eun
JE
Park
Seongsoon
S
Cheon
Yong-Pil
YP
Lim
Hyunjung Jade
HJ
D000584:Amiloride / Q000737:chemistry*; D000818:Animals; D015638:Cytochalasin D / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D034281:Dynamins / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009865:Oocytes / Q000378:metabolism; D021381:Protein Transport; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D047392:beta-Cyclodextrins / Q000737:chemistry
D004268:DNA-Binding Proteins; C092350:FM 4-64; C475074:MPG peptide; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; D047392:beta-Cyclodextrins; C485184:enhanced green fluorescent protein; C108732:methyl-beta-cyclodextrin; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D000584:Amiloride; D034281:Dynamins
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