Intracellular transport of rat serum albumin is altered by a genetically engineered deletion of the propeptide.
Many secreted proteins are synthesized with aminoterminal propeptides which are removed prior to secretion. There is increasing interest in the physiological roles of these propeptides, especially as mediators of intracellular protein trafficking. To investigate whether or not the propeptide of serum albumin offers an advantage in albumin secretion, we used oligonucleotide-directed mutagenesis to delete the 18 base pairs which encode the propeptide from a cDNA gene for rat serum albumin (RSA), inserted the deleted gene into COS cells, and studied the secretion of the gene product (RSA delta pro). Quantitative enzyme-linked immunosorbent assay analysis of medium from transfected cells showed that RSA delta pro was secreted at about 64% of the level of RSA. Furthermore, pulse-chase protein labeling studies demonstrated that the rate of secretion for RSA delta pro was greatly decreased relative to RSA. Immunofluorescent analyses of transfected cells showed accumulation of RSA delta pro in the endoplasmic reticulum, suggesting that transport through and/or exit from the ER was affected. The electrophoretic migration of secreted and intracellular forms of RSA and RSA delta pro indicated that they were the same molecular weight, and a specific amino-terminal binding assay, using nickel 63, confirmed the absence and proper cleavage of the prepeptide. These findings demonstrate that transport of RSA delta pro through the secretion pathway is inhibited and that the inhibition is due to the absence of the propeptide.
McCracken
A A
AA
Department of Biology, University of Nevada-Reno 89557.
Kruse
K B
KB
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005796:Genes; D005818:Genetic Engineering / Q000379:methods; D009154:Mutation; D009532:Nickel / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D011228:Prealbumin / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D012709:Serum Albumin / Q000378:metabolism*; D014162:Transfection
D009692:Nucleic Acid Heteroduplexes; D011228:Prealbumin; D012709:Serum Albumin; D009532:Nickel; D004247:DNA