Correct localization of Rab GTPases in cells is critical for proper function in membrane trafficking. Guanine-nucleotide exchange factors (GEFs) act as the primary determinants of Rab localization by activating and stabilizing their Rab substrates on specific organelle and vesicle membranes. The TRAPP complexes TRAPPII and TRAPPIII are two related GEFs that use the same catalytic site to activate distinct Rabs, Rab11 and Rab1, respectively. The Rab C-terminal hypervariable domain (HVD) is an important specificity determinant for the budding yeast TRAPP complexes, with the length of the HVD playing a critical role in counter-selection. Several recent studies have used cryo-EM to illuminate how the yeast and metazoan TRAPP complexes identify and activate their substrates. This review summarizes recently characterized Rab substrate selection mechanisms and highlights how the membrane surface provides critical context for the GEF-GTPase interactions.

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.

Yeast Sec14-like phosphatidylinositol transfer proteins (PITPs) contain a hydrophobic cavity capable of accepting a single molecule of phosphatidylinositol (PI) or another molecule in a mutually exclusive manner. We report here that two yeast Sec14 family PITPs, Pdr16p (Sfh3p) and Pdr17p (Sfh4p), possess high-affinity binding and transfer towards lanosterol. To our knowledge, this is the first identification of lanosterol transfer proteins. In addition, a pdr16Δpdr17Δ double mutant had a significantly increased level of cellular lanosterol compared with the corresponding wild-type. Based on the lipid profiles of wild-type and pdr16Δpdr17Δ cells grown in aerobic and anaerobic conditions, we suggest that PI-lanosterol transfer proteins are important predominantly for the optimal functioning of the post-lanosterol part of sterol biosynthesis.

The Transport Protein Particle (TRAPP) complexes are highly conserved multisubunit complexes that act as nucleotide exchange factors (GEFs) for Rab GTPases. They act in both protein secretion and autophagy and have also been proposed to have a role in other processes such as cytokinesis and ciliogenesis. There are two TRAPP complexes in metazoans: TRAPPII, which activates Rab11; and TRAPPIII, which activates Rab1. Both complexes share a core of small subunits that form the active site for the exchange of GDP for GTP. In addition, each TRAPP complex has distinct large subunits that determine the specificity of each complex towards its substrate Rab and are essential for activity in vivo. Crystal structures have revealed the organisation of the TRAPP core and the mechanism of Rab1 activation, whilst recent cryo-EM structures have unveiled the arrangement of the specific subunits around the core to form each complex. Combining these findings with functional experiments has allowed the proposal of mechanisms for how the specificity of each complex towards their cognate Rab is determined and for the arrangement of these large complexes on the membrane.

OGFOD1, a prolyl-hydroxylase, has been reported to translocate from the nucleus to the cytoplasm in response to cellular stress. Here, we demonstrate that OGFOD1 regulates the transcription and post-transcriptional stabilization of cell cycle-related genes. OGFOD1 knockdown in lung cancer cells induced cell cycle arrest through the specific depletion of cyclin-dependent kinase (CDK) 1, CDK2 and cyclin B1 (CCNB1) mRNAs and the nuclear accumulation of p21Cip1 . Analysis of the mRNA dynamics in these cells revealed that CDK1 decreased in a time-dependent manner, reflecting post-transcriptional regulation by OGFOD1 and the RNA-binding protein HuR. In contrast, the depletion of CDK2 and CCNB1 resulted from decreased transcription mediated by OGFOD1. These results indicate that OGFOD1 is required to maintain the function of specific cell cycle regulators during cancer cell proliferation.

Cytochrome bd-I from Escherichia coli is a terminal oxidase in the respiratory chain that plays an important role under stress conditions. Cytochrome bd-I was thought to consist of the major subunits CydA and CydB plus the small CydX subunit. Recent high-resolution structures of cytochrome bd-I demonstrated the presence of an additional subunit, CydH/CydY (called CydH here), the function of which is unclear. In this report, we show that in the absence of CydH, cytochrome bd-I is catalytically active, can sustain bacterial growth and displays haem spectra and susceptibility for haem-binding inhibitors comparable to the wild-type enzyme. Removal of CydH did not elicit catalase activity of cytochrome bd-I in our experimental system. Taken together, in the absence of the CydH subunit cytochrome bd-I retained key enzymatic properties.

Antifreeze proteins (AFPs) protect organisms from freezing by binding to ice crystals to prevent their growth. Here, we have investigated how the area of an AFP's ice-binding site (IBS) changes its antifreeze activity. The polyproline type II helical bundle fold of the 9.6-kDa springtail (Collembola) AFP from Granisotoma rainieri (a primitive arthropod) facilitates changes to both IBS length and width. A one quarter decrease in area reduced activity to less than 10%. A one quarter increase in IBS width, through the addition of a single helix, tripled antifreeze activity. However, increasing IBS length by a similar amount actually reduced activity. Expanding the IBS area can greatly increase antifreeze activity but needs to be evaluated by experimentation on a case-by-case basis.

During mitosis in metazoan species, the nuclear envelope (NE) undergoes breakdown, and its fragments are absorbed within the membranous network of the endoplasmic reticulum (ER). Past observations by fluorescence microscopy led researchers to think that the NE loses its identity when it is absorbed within the ER membrane. However, in our previous work, we developed a more specific labelling method and found evidence that the NE does not completely lose its identity during mitosis. In the present work, we conduct further experiments, the results of which support the idea that the NE partially retains its identity during mitosis.

Motile cells have developed a large array of molecular machineries to actively change their direction of movement in response to spatial cues from their environment. In this process, small GTPases act as molecular switches and work in tandem with regulators and sensors of their guanine nucleotide status (GAP, GEF, GDI and effectors) to dynamically polarize the cell and regulate its motility. In this review, we focus on Myxococcus xanthus as a model organism to elucidate the function of an atypical small Ras GTPase system in the control of directed cell motility. M. xanthus cells direct their motility by reversing their direction of movement through a mechanism involving the redirection of the motility apparatus to the opposite cell pole. The reversal frequency of moving M. xanthus cells is controlled by modular and interconnected protein networks linking the chemosensory-like frizzy (Frz) pathway - that transmits environmental signals - to the downstream Ras-like Mgl polarity control system - that comprises the Ras-like MglA GTPase protein and its regulators. Here, we discuss how variations in the GTPase interactome landscape underlie single-cell decisions and consequently, multicellular patterns.

Human growth hormone (hGH) is a four-helix bundle protein of considerable pharmacological interest. Recombinant hGH is produced in bacteria, yet little is known about its folding during expression in Escherichia coli. We have studied the cotranslational folding of hGH using force profile analysis (FPA), both during in vitro translation in the absence and presence of the chaperone trigger factor (TF), and when expressed in E. coli. We find that the main folding transition starts before hGH is completely released from the ribosome, and that it can interact with TF and possibly other chaperones.

Glucokinase (GCK) is the pancreatic β-cell glucose sensor, and its kinetics are key to that purpose. A slow transition step, displayed as non-hyperbolic kinetics, and a low affinity for glucose characterize GCK. Mutations in GCK associated with maturity-onset diabetes of the young type 2 (MODY2) previously described reduce the functionality of the human pancreatic β-cell, leading to diabetic clinical phenotypes. We present a kinetic characterization of the G448D mutation identified in a MODY2 patient, which is one of the first mutations to exhibit increased functionality. This mutant displays increased activity, high affinity for both Mg2+ -ATP and glucose, hyperbolic kinetics and increased phosphorylation potential. Hyperbolic kinetics and assays in the presence of glycerol indicate that G448D lacks the slow transition step crucial for the pancreatic β-cell glucose sensor function.

COPI-coated vesicles mediate transport between Golgi stacks and retrograde transport from the Golgi to the endoplasmic reticulum. The COPI coat exists as a stable heptameric complex in the cytosol termed coatomer and is recruited en bloc to the membrane for vesicle formation. Recruitment of COPI onto membranes is mediated by the Arf family of small GTPases, which, in their GTP-bound state, bind both membrane and coatomer. Arf GTPases also influence cargo selection, vesicle scission and vesicle uncoating. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) regulate nucleotide binding by Arf GTPases. To understand the mechanism of COPI-coated vesicle trafficking, it is necessary to characterize the interplay between coatomer and Arf GTPases and their effectors. It is also necessary to understand interactions between coatomer and cargo, cargo adaptors/receptors and tethers facilitating binding to the target membrane. Here, we summarize current knowledge of COPI coat protein structure; we describe how structural and biochemical studies contributed to this knowledge; we review mechanistic insights into COPI vesicle biogenesis and disassembly; and we discuss the potential to answer open questions in the field.