SV40 Large T-antigen nuclear signal analogues: Successful nuclear targeting with bovine serum albumin but not low molecular weight fluorescent conjugates
✍ Scribed by Thomas J. Lobl; Mark A. Mitchell; Linda L. Maggiora
- Book ID
- 102763564
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 1990
- Tongue
- English
- Weight
- 621 KB
- Volume
- 29
- Category
- Article
- ISSN
- 0006-3525
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✦ Synopsis
The signal sequence of a nuclear-directed protein encodes the necessary information for targeting the attached proteins to the cell nucleus. The sequence/structural requirements for a functional transport signal were explored with a series of peptides derived from the simian virus 40 large T-antigen nuclear signal 126-134 (CPKKKRKVED-NH2, wild type) conjugated to bovine serum albumin (BSA) through an N-terminal Cys ( 1 ) with mmaleimidobenzoyl-N-hydroxysuccinimide ester. Nuclear accumulation was virtually complete 15 min after microinjection into green monkey kidney cells (TC-?).
Peptides with Asn, Om, and Gln substituted for L~S'~', the reverse wild-type peptide (DEVKRKKKPC-NH2) and the long 34-residue wild-type analogue (CYDDEATADSQHSTPPKKKRKVEDPKDFESELLSNH2), were synthesized and conjugated similarly to BSA. The Orn peptide and the 34-residue wild-type analogue conjugated to ESA also transported to the nucleus but at a slower rate than 1. The reverse wild-type, Asn-and Gln-BSA conjugates of these signal analogues did not show transport to the nucleus after 6 h of incubation. In an effort to learn if such signal sequences would also target a small molecule such as a fluorescent tag to the nucleus, 1 fluorescently tagged with monobromobimane was prepared and microinjected into TC-7 cells. The peptide was distributed throughout the cell. These results support the notion that a positively charged residue at position 128 is needed for rapid nuclear transport and that the intracellular transport machinery has spatial recognition. The results with fluorophore-peptide conjugates suggest nuclear localization of these low molecular weight peptides will be difficult to attain even if attached to a functional nuclear localization sequence.