Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) Many biological processes are orchestrated by imposing spatial and temporal regulation on interactions between proteins. Therefore, identification of post-translational modifications and interaction networks among proteins is an essential aspect of modern biology. The tandem affinity purification (TAP) 1 method, originally developed for isolation of native protein complexes in yeast (1), allowed for diverse proteins to be easily purified using a single purification scheme. The TAP method works through expression of a fusion protein consisting of the TAP tag and the bait protein. The TAP tag contains two domains and motifs for tandem affinity purification steps; the rationale being that the use of the second step removes common contaminants that might be specific for the first step. Isolated protein complexes can then be used for biochemical experiments or analyzed by liquid-chromatography-tandem mass-spectrometry (LC-MS/MS). The TAP method has been transferred to mammalian cells; however, the overall recovery of native complexes is low (2). Several modified TAP methods have been developed for mammalian cells, using various combinations of affinity tags. Some tags work well for native protein complexes (2-4), and others for purification under denaturing conditions (5). Other TAP systems incorporate fluorescent proteins to allow for protein localization and to facilitate FACS of individual cells expressing various levels of the tagged protein (6, 7). Each of these purification tags has strengths and weaknesses. Here we describe a fluorescent protein-based multifunctional affinity purification (MAP) tag that incorporates the advantages of multiple purification systems into a single tag. We show that the MAP tag allows protein visualization, rapid selection of cell lines, and efficient affinity purification for all proteins tested under either native or denaturing conditions in mammalian cells, Caenorhabditis elegans, and the fission yeast, Schizosaccharomyces pombe. EXPERIMENTAL PROCEDURESPlasmids-Fluorescent proteins EGFP, sfGFP, mVenus, mCherry, and mKate2 were modified by insertion of the His 8 -SBP-FLAG From the ‡Department

In vivo biosensors that can convert metabolite concentrations into measurable output signals are valuable tools for high-throughput screening and dynamic pathway control in the field of metabolic engineering. Here, we present a novel biosensor in Saccharomyces cerevisiae that is responsive to p-coumaroyl-CoA, a central precursor of many flavonoids. The sensor is based on the transcriptional repressor CouR from Rhodopseudomonas palustris and was applied in combination with a previously developed malonyl-CoA biosensor for dual regulation of p-coumaroyl-CoA synthesis within the naringenin production pathway. Using this approach, we obtained a naringenin titer of 47.3 mg/L upon external precursor feeding, representing a 15-fold increase over the nonregulated system.

Structural Maintenance of Chromosomes (SMC) family proteins participate in multisubunit complexes that govern chromosome structure and dynamics. SMC-containing condensin complexes create chromosome topologies essential for mitosis/meiosis, gene expression, recombination, and repair. Many eukaryotes have two condensin complexes (I and II); C. elegans has three (I, II, and the X-chromosome specialized condensin IDC) and their regulation is poorly understood. Here we identify a novel SMC-like protein, SMCL-1, that binds to C. elegans condensin SMC subunits, and modulates condensin functions. Consistent with a possible role as a negative regulator, loss of SMCL-1 partially rescued the lethal and sterile phenotypes of a hypomorphic condensin mutant, while over-expression of SMCL-1 caused lethality, chromosome mis-segregation, and disruption of condensin IDC localization on X chromosomes. Unlike canonical SMC proteins, SMCL-1 lacks hinge and coil domains, and its ATPase domain lacks conserved amino acids required for ATP hydrolysis, leading to the speculation that it may inhibit condensin ATPase activity. SMCL-1 homologs are apparent only in the subset of Caenorhabditis species in which the condensin I and II subunit SMC-4 duplicated to create the condensin IDC- specific subunit DPY-27, suggesting that SMCL-1 helps this lineage cope with the regulatory challenges imposed by evolution of a third condensin complex. Our findings uncover a new regulator of condensins and highlight how the duplication and divergence of SMC complex components in various lineages has created new proteins with diverse functions in chromosome dynamics.

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