Patricia Grob

Saccharomyces cerevisiae septins: Supramolecular organization of heterooligomers and the mechanism of filament assembly

Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. The four essential septins form a complex containing two copies of each, but their arrangement was not known. Single-particle analysis by EM confirmed that the heterooligomer is octameric and revealed that the subunits are arrayed in a linear rod. Identity of each subunit was determined by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (GFP or maltose binding protein). The rod has the order Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end-to-end to form filaments but not when Cdc11 is absent or its N terminus is altered. Filaments invariably pair into long parallel “railroad tracks.” Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Shs1 may be able to replace Cdc11 at the end of the rod. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures.

Modularity of a carbon-fixing protein organelle

Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO2-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO2. Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO2 in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.

In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release

Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca2+ triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca2+ injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca2+ injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca2+ sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca2+ triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control.

PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE PROMOTES BUDDING YEAST SEPTIN FILAMENT ASSEMBLY AND ORGANIZATION

Septins are a conserved family of GTP-binding proteins that assemble into symmetric linear heterooligomeric complexes, which in turn are able to polymerize into apolar filaments and higher-order structures. In budding yeast (Saccharomyces cerevisiae) and other eukaryotes, proper septin organization is essential for processes that involve membrane remodeling, such as the execution of cytokinesis. In yeast, four septin subunits form a Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 heterooctameric rod that polymerizes into filaments thought to form a collar around the bud neck in close contact with the inner surface of the plasma membrane. To explore septin-membrane interactions, we examined the effect of lipid monolayers on septin organization at the ultrastructural level using electron microscopy. Using this methodology, we have acquired new insights into the potential effect of septin-membrane interactions on filament assembly and, more specifically, on the role of phosphoinositides. Our studies demonstrate that budding yeast septins interact specifically with phosphatidylinositol-4,5-bisphosphate (PIP2) and indicate that the N terminus of Cdc10 makes a major contribution to the interaction of septin filaments with PIP2. Furthermore, we found that the presence of PIP2 promotes filament polymerization and organization on monolayers, even under conditions that prevent filament formation in solution or for mutants that prevent filament formation in solution. In the extreme case of septin complexes lacking the normally terminal subunit Cdc11 or the normally central Cdc10 doublet, the combination of the PIP2-containing monolayer and nucleotide permitted filament formation in vitro via atypical Cdc12-Cdc12 and Cdc3-Cdc3 interactions, respectively.

Synthesis of Heterobifunctional Protein Fusions Using Copper-Free Click Chemistry and the Aldehyde Tag

Heterobifunctional protein fusions are gaining interest as next-generation biopharmaceuticals.1–5 Combining proteins with disparate functions can enable multidrug therapy with a single chemical entity,6, 7 add a targeting element to an otherwise nonspecific therapeutic,8, 9 or improve the pharmacokinetic profile of a rapidly cleared molecule.10, 11 Indeed, heterobifunctional proteins, such as immunoglobulin G (IgG) Fc domain fusions, are among the top-selling biotherapeutics on the market today.12 These biomolecules are primarily generated as genetic fusions. The DNA sequences that encode the individual protein components are fused in tandem to direct the expression of a single polypeptide that comprises the two proteins joined together at their N and C termini, respectively. However, this limited topology is not ideal for every protein combination, as some polypeptides require unmodified termini for optimal bioactivity13 or can suffer from expression difficulties as a result of folding and processing issues.3, 14, 15

Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways. DOI: http://dx.doi.org/10.7554/eLife.00109.001

Human TFIID Binds to Core Promoter DNA in a Reorganized Structural State

A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single-particle electron microscopy (EM) to visualize human TFIID with promoter DNA. This analysis revealed that TFIID coexists in two predominant and distinct structural states that differ by a 100 Å translocation of TFIIDs lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a rearranged state of TFIID that enables promoter recognition and binding. DNA labeling and footprinting, together with cryo-EM studies, were used to map the locations of TATA, Initiator (Inr), motif ten element (MTE), and downstream core promoter element (DPE) promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors.

Molecular architecture of the human Mediator-RNA polymerase II-TFIIF assembly.

The authors perform a cryo-EM study of the 1.9 MDa human Mediator-RNA polymerase II-TFIIF assembly, which reveals the structural organization of the human transcription initiation apparatus.

Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. DOI: http://dx.doi.org/10.7554/eLife.05115.001

Structural Changes in TAF4b-TFIID Correlate with Promoter Selectivity

Proper ovarian development requires the cell type-specific transcription factor TAF4b, a subunit of the core promoter recognition complex TFIID. We present the 35 A structure of a cell type-specific core promoter recognition complex containing TAF4b and TAF4 (4b/4-IID), which is responsible for directing transcriptional synergy between c-Jun and Sp1 at a TAF4b target promoter. As a first step toward correlating potential structure/function relationships of the prototypic TFIID versus 4b/4-IID, we have compared their 3D structures by electron microscopy and single-particle reconstruction. These studies reveal that TAF4b incorporation into TFIID induces an open conformation at the lobe involved in TFIIA and putative activator interactions. Importantly, this open conformation correlates with differential activator-dependent transcription and promoter recognition by 4b/4-IID. By combining functional and structural analysis, we find that distinct localized structural changes in a megadalton macromolecular assembly can significantly alter its activity and lead to a TAF4b-induced reprogramming of promoter specificity.