SV40 large T-antigen nuclear signal analogues: successful nuclear targeting with bovine serum albumin but not low molecular weight fluorescent conjugates.
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 m-maleimidobenzoyl-N-hydroxysuccinimide ester. Nuclear accumulation was virtually complete 15 min after microinjection into green monkey kidney cells (TC-7). Peptides with Asn, Orn, and Gln substituted for Lys128, the reverse wild-type peptide (DEVKRKKPC-NH2) and the long 34-residue wild-type analogue (CYDDEATADSQHSTPPKKKRKVEDPKDFESELLS-NH2), were synthesized and conjugated similarly to BSA. The Orn peptide and the 34-residue wild-type analogue conjugated to BSA 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.
Lobl
T J
TJ
Biopolymer Chemistry Research, Upjohn Company, Kalamazoo, Michigan 49001.
Mitchell
M A
MA
Maggiora
L L
LL
D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming; D002467:Cell Nucleus / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000138:chemical synthesis / Q000378:metabolism*; D012710:Serum Albumin, Bovine
D000952:Antigens, Polyomavirus Transforming; D005456:Fluorescent Dyes; D021382:Protein Sorting Signals; D012710:Serum Albumin, Bovine