Subcellular Protein Localization by Using a Genetically Encoded Fluorescent Amino Acid

The use of fluorescent protein fusions has revolutionized cell biology by allowing exploration of proteins in their native context. However, the utilization of current techniques is limited by the size and placement of the fused fluorescent protein. This is especially true for proteins that oligomerize or assemble into large complexes in which the fluorescent protein fusion can lead to improper assembly and/or function. To circumvent this problem, we report a novel technique for fluorescent labeling of proteins, in vivo. The unique potential of this technique lies in the ability to place a very small fluorescent tag virtually anywhere along a chosen protein sequence, thereby minimizing the risk of affecting protein function. To illustrate the utility of this method, we have genetically encoded a single unnatural fluorescent amino acid in the sequence of the bacterial tubulin, FtsZ. This resulted in the production of a functional protein that could be visualized, in vivo. The aim of this study was to explore novel methods of labeling proteins for in vivo localization studies that do not perturb protein function or structure. To this end, we have exploited a technique that allows unnatural amino acids with novel properties, in our case a fluorescent, coumarin-derived amino acid (CouAA; Figure 1A), to be encoded into a given sequence. The technique uses an orthogonal tRNA/aminoacyl-tRNA synthetase pair that transfers a defined unnatural amino acid to a growing polypeptide chain when nonsense amber codons (UAG) are present in the coding sequence. Specifically, we have used an archaebacteria amber suppressor tRNA (MjtRNA)/aminoacyl-tRNA synthetase (Mj-aaRS) pair (from Methanococcus jannaschi) that does not interact with endogenous E. coli aminoacyl-tRNA synthetases or tRNAs. This aaRS (CouRS) was then evolved by using a two-step selection process to specifically recognize CouAA and not an endogenous host amino acid. As a consequence, by simply inserting an amber stop codon in the desired gene sequence, an unnatural amino acid will be introduced in the corresponding translated protein (see cartoon in the Supporting Information). Conveniently, endogenous amber codons in E. coli and other organisms are likely poorly recognized by the Mj-tRNA, so that the expression of genes terminated by amber codons is apparently not affected. Although the exact mechanisms are not understood, it is known that the termination and suppression processes are influenced by sequences adjacent to the amber codon. To demonstrate that this system can be effectively used as a means to visualize the in vivo subcellular location of a CouAAlabeled protein in bacteria, we chose to label the bacterial tubulin homologue FtsZ, which has been extensively studied both in vitro and in vivo. FtsZ assembles into a contractile ring (visible by fluorescence microscopy as a midcell band called Zring) during cytokinesis. But fusion of FtsZ to a fluorescent protein impairs its cellular function, a well-known problem for cytoskeletal proteins. Consequently, to date, all the reported FtsZ–fluorescent protein fusions have been shown to be nonfunctional on their own and must be produced in the presence of an untagged FtsZ copy for normal cell function. Thus, in this context, the FtsZ–fluorescent protein fusions merely label the endogenous, untagged FtsZ structure. While this approach has proven to be sufficient to generate considerable insight into FtsZ cellular function, it remains an imperfect artifice that might fail in the case of other oligomer-forming proteins or proteins forming macromolecular complexes. To visualize FtsZ in E. coli, we substituted the tenth amino acid (Asp10) for CouAA (FtsZ10CouAA). Asp10 is located in a disordered N-terminal segment with no known function and is likely to be surface exposed. To characterize the specificity of CouAA incorporation, Histagged FtsZ10CouAA was expressed in BL21(DE3) E. coli cells transformed with two plasmids: one constitutively expressed the evolved Mj-tRNA (pBKcouRS) and the other one (pBADJYftsZH6D10TAG) constitutively expressed the evolved tRNA synthetase (CouRS) and the mutated ftsZ gene under an arabinose inducible promoter. SDS-PAGE analysis (Figure 1B) of total cell extract of cells grown in the absence or presence of CouAA (1 mm) in the growth medium to express FtsZ10CouAA, showed a unique band at approximately 40 kDa only with [a] Dr. G. Charbon, Prof. Dr. C. Jacobs-Wagner Department of Molecular, Cellular, and Developmental Biology KBT 1032, Yale University, New Haven, CT 06520 (USA) [b] Dr. G. Charbon, Prof. Dr. A. Løbner-Olesen Department of Science, Systems and Models, Roskilde University Building 18.1, 4000 Roskilde (Denmark) [c] Dr. E. Brustad, Prof. Dr. J. Wang, Prof. Dr. P. G. Schultz Department of Chemistry, The Scripps Research Institute SR202, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA) [d] K. A. Scott, Prof. Dr. E. Chapman Department of Molecular Biology, The Scripps Research Institute MB46, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA) E-mail : [email protected] [e] Prof. Dr. C. Jacobs-Wagner Howard Hughes Medical Institute New Haven, CT 06510 (USA) [f] Prof. Dr. C. Jacobs-Wagner Microbial Pathogenesis Section, Yale School of Medicine New Haven, CT 06510 (USA) [g] Prof. Dr. J. Wang Present address: National Key Laboratory of Biomacromolecules Institute of Biophysics, Chinese Academy of Sciences Beijing 100101 (China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201100282.