Tus, an E. coli Protein, Contains Mammalian Nuclear Targeting and Exporting Signals

Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS) and nuclear export signals (NES), respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells

Protein Microarray On-Demand: A Novel Protein Microarray System

We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (∼3–7×10 −13 M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This “all DNA” microarray can be converted to a protein microarray on-demand without need for any additional capture reagent.

A. Location of NES sequence, comparison of various NES and mutational analysis of NES region of Tus.

<p>A. Replacement of full-length Tus in pDest 472 GFP-Tus vector with the indicated region of Tus was used to localize the region of NES. B. Comparison of Tus NES with known NES (p53, HIV Rev and PKI) were shown. Amino acids in red indicate leucine, and isoleucine residues are hallmarks of NES. C. Effect of mutation of leucine 33 and 34 in NES function. (a) Localization of GFP-Tus (1–77) and (b) GFP-Tus (1–77:L33A–L34A double mutants) in PC3 cells. D. Distribution of GFP proteins in PC3 cell line.</p

Subcellular localization of GFP and GFP-Tus derivatives.

<p>A Panel (a), GFP only; Panel (b), GFP-TUS is the fusion of GFP with full-length Tus; Panel (c), GFP-TUS-ΔC is GFP-fused with amino acids Tus containing 1–217 of Tus; (d), GFP-TetR is the fusion of GFP with a non specific protein, tetR. B. Panel (a), GFP fluorescence of GFP-Tus protein; Panel (b), cells were stained with DAPI; and Panel (c), Superimposition of panels (a) and (b). C. Cytoplasmic localization of GFP fused with 1–217 amino acids of Tus. Panel (a), GFP fluorescence; Panel (b), DAPI staining; and Panel (c), Superimposition of panels (a) and (b).</p

Protein delivery using GFP-Tus and GFP-Tus NLS fusion protein.

<p>A. Internalization of GFP-Tus fusion protein in PC3 cell line after 24 hrs post-addition of the fusion protein. B. Internalization of GFP-Tus NLS fusion protein after 2 hrs; or C. 24 hrs post-addition of the fusion protein. D. Addition of GFP fusion protein (control) showed no internalization (negative control).</p

A. Localization of NLS and NES determinants of Tus by deletion analysis.

<p>A. Full-length and various deletions of Tus were fused with GFP, and localization of the fusion protein in nucleus (N), cytoplasm (C), and all over the cell (C+N) were indicated on right. The boundary of the NLS region was between amino acids 218 and 264 and the boundary of the NES region was between 1 and 77 amino acids. B. Comparison of various types of known NLS with putative NLS sequence of Tus.</p