A new approach to an old problem: synthetic biology tools for human disease and metabolism.

The field of synthetic biology seeks to develop engineering principles for biological systems. Along these lines, synthetic biology can address human metabolic disease through the development of genetic approaches to the study and modification of metabolism. The re-engineering of natural metabolic states provides fundamental understanding of the integrated components underlying dysfunctional metabolism. Alternatively, the development of biological devices that can both sense and affect metabolic states could render unique control over disease states. In this chapter, we discuss the advancement of synthetic biological approaches to monitoring and engineering metabolism, as well as prospects for synthetic biology's future role in the prevention and treatment of metabolic disease.

Nuclear origins of cell-to-cell variability.

During the past decade, it has become increasingly evident that there is variation in the transcriptome of genetically identical cells, even when grown in homogenous environments. This cell-to-cell variability has been shown to have a central role in processes ranging from stem cell differentiation to chemotherapy resistance. Given that many genes display extensive heterogeneity in their messenger RNA (mRNA) abundance on a per cell basis, understanding the nuclear sources of this variability is important for our fundamental grasp of nuclear function and stands to have clinical manifestations. In this chapter, we assess the contribution of different transcription regimes, nuclear architecture dynamics, RNA polymerase elongation, and gene copy number to transcriptome heterogeneity. We also discuss techniques that can be used to quantify single-cell mRNA abundance and conclude by commenting on future research directions.

Molecular systems biology in drug development.

Molecular systems biology seeks to explain the behavior of complex cellular systems through a multicomponent analysis. We illustrate this approach and its relevance to drug development by reviewing two examples of the interplay between cellular processes and drugs: the internalization and recycling of oncogenic receptors, their ligands and therapeutic antibodies; and strategies for discovering drugs that affect intracellular protein kinase pathways.

The conserved npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation.

Proteolytic activation of membrane-bound transcription factors has emerged as an important mechanism for the regulation of gene expression. Two membrane-bound transcription factors regulated in this manner are the Saccharomyces cerevisiae proteins Mga2p and Spt23p, which direct transcription of the Delta9-fatty acid desaturase gene OLE1. We now show that a membrane-associated complex containing the highly conserved Npl4p, Ufd1p, and Cdc48p proteins mediates the proteasome-regulated cleavage of Mga2p and Spt23p. Mutations in NPL4, UFD1, and CDC48 cause a block in Mga2p and Spt23p processing, with concomitant loss of OLE1 expression. Taken together, our data indicate that the Npl4 complex may serve to target the proteasome to the ubiquitinated endoplasmic reticulum membrane-bound proteins Mga2p and Spt23p. Given the recent finding that NPL4 is allelic to the ERAD gene HRD4, we further propose that this NPL4 function extends to all endoplasmic reticulum-membrane-associated targets of the proteasome.

Localization of yeast telomeres to the nuclear periphery is separable from transcriptional repression and telomere stability functions.

The left telomere of Saccharomyces chromosome VII was often localized near the nuclear periphery, even in cells lacking the silencing proteins Sir3 or Hdf1. This association was lost in late mitotic cells and when transcription was induced through the telomeric tract. Although in silencing competent cells there was no correlation between the fraction of cells in which a telomeric gene was repressed and the fraction of cells in which it was localized to the periphery, no condition was found where the telomere was both silenced and away from the periphery. We conclude that localization of a telomere to the nuclear periphery is not sufficient for transcriptional repression nor does it affect the stability function of yeast telomeres.

Messenger RNAs are recruited for nuclear export during transcription.

Following transcription and processing, eukaryotic mRNAs are exported from the nucleus to the cytoplasm for translation. Here we present evidence that mRNAs are targeted for nuclear export cotranscriptionally. Combined mutations in the Saccharomyces cerevisiae hnRNP Npl3 and TATA-binding protein (TBP) block mRNA export, implying that cotranscriptional recruitment of Npl3 is required for efficient export of mRNA. Furthermore, Npl3 can be found in a complex with RNA Pol II, indicating that Npl3 associates with the transcription machinery. Finally, Npl3 is recruited to genes in a transcription dependent manner as determined by chromatin immunoprecipitation. Another mRNA export factor, Yra1, also associates with chromatin cotranscriptionally but appears to be recruited at a later step. Taken together, our results suggest that export factors are recruited to the sites of transcription to promote efficient mRNA export.

The Saccharomyces cerevisiae cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting determinants.

The cyclin-dependent kinase Cdc28p associates with the cyclin Clb2p to induce mitosis in the yeast Saccharomyces cerevisiae. Several cell cycle regulatory proteins have been shown to require specific nuclear transport events to exert their regulatory functions. Therefore, we investigated the subcellular localization of wild-type Clb2p and several mutant versions of the protein using green fluorescent protein (GFP) fusion constructs. Wild-type Clb2p is primarily nuclear at all points of the cell. A point mutation in a potential leucine-rich nuclear export signal (NES) enhances the nuclear localization of the protein, and delta-yrb2 cells exhibit an apparent Clb2p nuclear export defect. Clb2p contains a bipartite nuclear localization signal (NLS), and its nuclear localization requires the alpha and beta importins (Srp1p and Kap95p), as well as the yeast Ran GTPase and its regulators. Deletion of the Clb2p NLS causes increased cytoplasmic localization of the protein, as well as accumulation at the bud neck. These data indicate that Clb2p exists in multiple places in the yeast cell, possibly allowing Cdc28p to locally phosphorylate substrates at distinct subcellular sites.

Use of the A. victoria green fluorescent protein to study protein dynamics in vivo.

Fluorescent molecules serve as valuable tools for the detection of numerous biochemical phenomena and have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting and determination of chemical concentrations. However, the use of such techniques generally requires significant nonphysiological perturbations to the biological system being studied; therefore, they are not always appropriate for the observation of dynamic phenomena. Green fluorescent protein (GFP), cloned from jellyfish, has been used to overcome many of these problems. It is a small, extremely stable fluorescent protein that has been successfully expressed and detected in a wide variety of organisms, both in intact form and fused to other proteins. This overview unit describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena.

Use of the A. victoria green fluorescent protein to study protein dynamics in vivo.

Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has heretofore been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.

Use of the A. victoria green fluorescent protein to study protein dynamics in vivo.

Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.

The structure and oligomerization of the yeast arginine methyltransferase, Hmt1.

Protein methylation at arginines is ubiquitous in eukaryotes and affects signal transduction, gene expression and protein sorting. Hmt1/Rmt1, the major arginine methyltransferase in yeast, catalyzes methylation of arginine residues in several mRNA-binding proteins and facilitates their export from the nucleus. We now report the crystal structure of Hmt1 at 2.9 A resolution. Hmt1 forms a hexamer with approximate 32 symmetry. The surface of the oligomer is dominated by large acidic cavities at the dimer interfaces. Mutation of dimer contact sites eliminates activity of Hmt1 both in vivo and in vitro. Mutating residues in the acidic cavity significantly reduces binding and methylation of the substrate Npl3.

Factors affecting nuclear export of the 60S ribosomal subunit in vivo.

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the approximately 45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b-GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b-GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b-GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b-GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120.

Mutants affecting the structure of the cortical endoplasmic reticulum in Saccharomyces cerevisiae.

We find that the peripheral ER in Saccharomyces cerevisiae forms a dynamic network of interconnecting membrane tubules throughout the cell cycle, similar to the ER in higher eukaryotes. Maintenance of this network does not require microtubule or actin filaments, but its dynamic behavior is largely dependent on the actin cytoskeleton. We isolated three conditional mutants that disrupt peripheral ER structure. One has a mutation in a component of the COPI coat complex, which is required for vesicle budding. This mutant has a partial defect in ER segregation into daughter cells and disorganized ER in mother cells. A similar phenotype was found in other mutants with defects in vesicular trafficking between ER and Golgi complex, but not in mutants blocked at later steps in the secretory pathway. The other two mutants found in the screen have defects in the signal recognition particle (SRP) receptor. This receptor, along with SRP, targets ribosome-nascent chain complexes to the ER membrane for protein translocation. A conditional mutation in SRP also disrupts ER structure, but other mutants with translocation defects do not. We also demonstrate that, both in wild-type and mutant cells, the ER and mitochondria partially coalign, and that mutations that disrupt ER structure also affect mitochondrial structure. Our data suggest that both trafficking between the ER and Golgi complex and ribosome targeting are important for maintaining ER structure, and that proper ER structure may be required to maintain mitochondrial structure.

7The yeast mRNA-binding protein Npl3p interacts with the cap-binding complex.

A number of RNA-binding proteins are associated with mRNAs in both the nucleus and the cytoplasm. One of these, Npl3p, is a heterogeneous nuclear ribonucleoprotein-like protein with some similarity to SR proteins and is essential for growth in the yeast S. cerevisiae. Temperature-sensitive alleles have defects in the export of mRNA out of the nucleus (1). In this report, we define a genetic relationship between NPL3 and the nonessential genes encoding the subunits of the cap-binding complex (CBP80 and CBP20). Deletion of CBP80 or CBP20 in combination with certain temperature-sensitive npl3 mutant alleles fail to grow and thus display a synthetic lethal relationship. Further evidence of an interaction between Npl3p and the cap-binding complex was revealed by co-immunoprecipitation experiments; Cbp80p and Cbp20p specifically co-precipitate with Npl3p. However, the interaction of Npl3p with Cbp80p depends on both the presence of Cbp20p and RNA. In addition, we show that Cbp80p is capable of shuttling between the nucleus and the cytoplasm in a manner dependent on the ongoing synthesis of RNA. Taken together, these data support a model whereby mRNAs are co-transcriptionally packaged by proteins including Npl3p and cap-binding complex for export out of the nucleus.

Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex.

The interactions between transport receptors and proteins of the nuclear pore complex (NPC) are fundamental to understanding nucleocytoplasmic transport. In order to delineate the path that a particular transport receptor takes through the NPC, we have employed fluorescence resonance energy transfer (FRET) between enhanced cyan and yellow fluorescent proteins (ECFP, EYFP) in living cells. A panel of yeast strains expressing functional receptor--ECFP and nucleoporin--EYFP fusions has been analyzed with a FRET assay. With this approach, we define points of contact in the NPC for the related importin Pse1/Kap121 and exportin Msn5. These data demonstrate the utility of FRET in mapping dynamic protein interactions in a genetic system. Furthermore, the data indicate that an importin and exportin have overlapping pathways through the NPC.

Nup2p is located on the nuclear side of the nuclear pore complex and coordinates Srp1p/importin-alpha export.

Proteins bearing canonical nuclear localization sequences are imported into the nucleus by the importin/karyopherin-alpha/beta heterodimer. Recycling of the importin-alpha subunit to the cytoplasm requires the action of Cas, a member of the importin-beta superfamily. In the yeast Saccharomyces ceresivisiae, the essential gene CSE1 encodes a Cas homologue that exports the yeast importin-alpha protein Srp1p/Kap60p from the nucleus. In this report, we describe a role for the FXFG nucleoporin Nup2p, and possibly the related Nup1p, in the Cse1p-mediated nuclear export pathway. Yeast cells lacking Nup2p or containing a particular temperature-sensitive mutation in NUP1 accumulate Srp1p in the nucleus. Similarly, Cse1p is displaced from the nuclear rim to the nuclear interior in deltanup2 cells. We do not observe any biochemical interaction between Cse1p and Nup2p. Instead, we find that Nup2p binds directly to Srp1p. We have localized Nup2p to the interior face of the nuclear pore complex, and have shown that its N terminus is sufficient for targeting Nup2p to the pore, as well as for binding to Srp1p. Taken together, these data suggest that Nup2p is an important NPC docking site in the Srp1p export pathway.