The adenovirus E1A nuclear localization signal: Analysis of sequence requirements and function.

摘要

Nucleocytoplasmic transport is a crucial component of the cellular machinery because it provides a link between the genetic information in the nucleus and protein synthesis in the cytoplasm. Understanding the mechanism(s) behind nuclear translocation of proteins and how this process is regulated is a fundamental problem in cell biology. Most nuclear proteins have a cluster of basic amino acids that serves as a nuclear localization signal (NLS). To learn more about NLS structure, point mutations in the NLS of the adenovirus E1a protein were produced. Fifteen signal sequence variants were tested and ranked according to their signal strength. Conclusions are based on comparisons of E1a NLS point mutants in which signal context was unchanged. Most of the E1a NLS (;The translocation of protein to the nucleus is also affected by parameters other than NLS sequence. Within the normal population of Vero cells, the presence of unusually responding cells in the cell culture population was also observed. Two to five percent of the cells exhibited rapid nuclear localization (5 vs. 30 min) or failed to localize E1a by 30 minutes post injection. To determine whether these cells are representative of different subpopulations of cells or arise spontaneously, twelve cloned cell lines were established from the parental Vero cell line. These clones were tested with the adenovirus E1a microinjection assay. All twelve cloned cell lines displayed the same heterogeneous response at 5 and 30 minutes post injection as the parental Vero cell line. Thus, because a minority of unusual cells at the 5 and 30 minute timepoints could be observed amongst a majority of typically responding cells, we conclude that this heterogeneity of response is an intrinsic characteristic of the cell line.