The effect of carboxyl-terminal deletions on the nuclear transport rate of rat hsc70.
Rat brain hsc70 is a constitutively expressed member of the 70-kDa family of heat shock proteins that is capable of bidirectional transport across the nuclear envelope when microinjected into Xenopus oocytes [1]. The objective of this study was to identify domains involved in its bidirectional transport. Limited proteolytic digestion with chymotrypsin generated three major truncated proteins of approximately 67.5, 59.5, and 56.5 kDa. Reactivity with NH2-terminal-specific antibodies showed that carboxyl-terminal fragments were removed. Nuclear uptake studies were performed by microinjecting 125I-labeled proteins into the cytoplasm and determining their subsequent nucleocytoplasmic distribution. The accumulation rates, while faster than bovine serum albumin controls, were inversely related to the size of the truncated proteins and greatly reduced compared to undigested hsc70. Nuclear efflux was assayed by microinjecting labeled proteins directly into oocyte nuclei. The relative efflux rates of the truncated polypeptides were less than the undigested protein, and, as observed for uptake, were inversely related to size. These results indicate that the carboxyl-terminal domain of hsc70 is involved in its bidirectional exchange.
Mandell
R B
RB
Department of Anatomy and Cell Biology, University of Florida, Gainesville 32610.
Feldherr
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D015151:Immunoblotting; D007700:Kinetics; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D014982:Xenopus laevis
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C494373:Hspa8 protein, rat; D002918:Chymotrypsin