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Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year.sup.1. Specialized protein transport systems--known as type VII secretion systems (T7SSs)--are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope.sup.2,3. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP.sub.5 protease. A trimer of MycP.sub.5 caps a central periplasmic dome-like chamber that is formed by three EccB.sub.5 dimers, with the proteolytic sites of MycP.sub.5 facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP.sub.5 show disruption of the EccB.sub.5 periplasmic assembly and increased flexibility, which highlights the importance of MycP.sub.5 for complex integrity. Beneath the EccB.sub.5-MycP.sub.5 chamber, dimers of the EccC.sub.5 ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC.sub.5 domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen. A cryo-electron microscopy structure of the inner membrane complex of the ESX-5 type VII secretion system of Mycobacterium tuberculosis reveals an important role of interactions with MycP.sub.5 protease for complex integrity., Author(s): Catalin M. Bunduc [sup.1] [sup.2] [sup.3] [sup.4] , Dirk Fahrenkamp [sup.1] [sup.2] [sup.3] , Jiri Wald [sup.1] [sup.2] [sup.3] , Roy Ummels [sup.5] , Wilbert Bitter [sup.4] [sup.5] , [...]

Oncogenic activation of RAS is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumour cell fitness. Cells that express oncogenic RAS are able to internalize and degrade extracellular protein via a fluid-phase uptake mechanism termed macropinocytosis.sup.1. There is increasing recognition of the role of this RAS-dependent process in the generation of free amino acids that can be used to support tumour cell growth under nutrient-limiting conditions.sup.2. However, little is known about the molecular steps that mediate the induction of macropinocytosis by oncogenic RAS. Here we identify vacuolar ATPase (V-ATPase) as an essential regulator of RAS-induced macropinocytosis. Oncogenic RAS promotes the translocation of V-ATPase from intracellular membranes to the plasma membrane via a pathway that requires the activation of protein kinase A by a bicarbonate-dependent soluble adenylate cyclase. Accumulation of V-ATPase at the plasma membrane is necessary for the cholesterol-dependent plasma-membrane association of RAC1, a prerequisite for the stimulation of membrane ruffling and macropinocytosis. These observations establish a link between V-ATPase trafficking and nutrient supply by macropinocytosis that could be exploited to curtail the metabolic adaptation capacity of RAS-mutant tumour cells. A whole-genome short interfering RNA screen identified vacuolar ATPase as a regulator of macropinocytosis induced by oncogenic RAS, a key determinant of tumour cell growth under nutrient-limiting conditions., Author(s): Craig Ramirez [sup.1] , Andrew D. Hauser [sup.1] , Emily A. Vucic [sup.1] , Dafna Bar-Sagi [sup.1] Author Affiliations: (1) Department of Biochemistry and Molecular Pharmacology, New York University [...]

Author(s): Qingfeng Chen [1, 2, 3]; Ji She [1, 2]; Weizhong Zeng [1, 2, 3]; Jiangtao Guo [1, 2]; Haoxing Xu [4]; Xiao-chen Bai (corresponding author) [2]; Youxing Jiang (corresponding [...]

Transient receptor potential mucolipin 1 (TRPML1) is a Ca[sup.2+]-releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type IV, a severe lysosomal storage disorder. Here we report two electron cryo-microscopy structures of full-length human TRPML1: a 3.72- apo structure at pH 7.0 in the closed state, and a 3.49- agonist-bound structure at pH 6.0 in an open state. Several aromatic and hydrophobic residues in pore helix 1, helices S5 and S6, and helix S6 of a neighbouring subunit, form a hydrophobic cavity to house the agonist, suggesting a distinct agonist-binding site from that found in TRPV1, a TRP channel from a different subfamily. The opening of TRPML1 is associated with distinct dilations of its lower gate together with a slight structural movement of pore helix 1. Our work reveals the regulatory mechanism of TRPML channels, facilitates better understanding of TRP channel activation, and provides insights into the molecular basis of mucolipidosis type IV pathogenesis., Author(s): Philip Schmiege [1, 2]; Michael Fine [3]; Gnter Blobel (corresponding author) [1]; Xiaochun Li (corresponding author) [1, 2, 4] TRPML1, a member of the mucolipin subfamily of transient receptor [...]

Author(s): Vctor Garca-Lpez [1, 2]; Fang Chen [3]; Lizanne G. Nilewski [1, 2]; Guillaume Duret [4]; Amir Aliyan [1]; Anatoly B. Kolomeisky [1]; Jacob T. Robinson (corresponding author) [4]; Gufeng [...]

Structural and functional studies of the tail knob protein of bacteriophage [phi]29 shed light on how the phage breaches the membrane barrier and ejects its DNA genome into the host cell. How the [phi]29 phage crosses the cell membrane Bacteriophages have to overcome several physical barriers in order to introduce their genome into the cytoplasm of the host cell. How they get across the inner bacterial membrane has been unclear. Here Ye Xiang and colleagues describe structural and functional studies of the bacteriophage [phi]29 tail knob protein. The structure reveals that six gp9 molecules form a hexameric tube structure with six flexible hydrophobic loops that form a channel spanning the lipid bilayer of the membrane for the release of bacteriophage genomic DNA. This points to a pore-forming mechanism for membrane penetration similar to that of certain non-enveloped eukaryotic viruses. Most bacteriophages are tailed bacteriophages with an isometric or a prolate head attached to a long contractile, long non-contractile, or short non-contractile tail.sup.1. The tail is a complex machine that plays a central role in host cell recognition and attachment, cell wall and membrane penetration, and viral genome ejection. The mechanisms involved in the penetration of the inner host cell membrane by bacteriophage tails are not well understood. Here we describe structural and functional studies of the bacteriophage [phi]29 tail knob protein gene product 9 (gp9). The 2.0 Å crystal structure of gp9 shows that six gp9 molecules form a hexameric tube structure with six flexible hydrophobic loops blocking one end of the tube before DNA ejection. Sequence and structural analyses suggest that the loops in the tube could be membrane active. Further biochemical assays and electron microscopy structural analyses show that the six hydrophobic loops in the tube exit upon DNA ejection and form a channel that spans the lipid bilayer of the membrane and allows the release of the bacteriophage genomic DNA, suggesting that cell membrane penetration involves a pore-forming mechanism similar to that of certain non-enveloped eukaryotic viruses.sup.2,3,4. A search of other phage tail proteins identified similar hydrophobic loops, which indicates that a common mechanism might be used for membrane penetration by prokaryotic viruses. These findings suggest that although prokaryotic and eukaryotic viruses use apparently very different mechanisms for infection, they have evolved similar mechanisms for breaching the cell membrane., Author(s): Jingwei Xu [sup.1] , Miao Gui [sup.1] , Dianhong Wang [sup.1] , Ye Xiang [sup.1] Author Affiliations: (1) Department of Basic Medical Sciences, Centre for Infectious Diseases Research, Collaborative [...]

The essential autophagy mediator ATG14 promotes vesicle fusion by forming homo-oligomers, which bind to a component of the SNARE membrane fusion complex and stabilize this complex on autophagosomes. Autophagy-specific membrane fusion During the process of autophagy, cells break down their own components and sequester them in specialized vesicles called autophagosomes, which eventually fuse with lysosomes for degradation of their content. Membrane fusion is an important event not only during early biogenesis of autophagosomal vesicles but also when autophagosomes unite with lysosomes. Qing Zhong and colleagues show here that the essential autophagy mediator ATG14 promotes vesicle fusion by forming homo-oligomers, which bind to a component of the SNARE membrane fusion complex and stabilize this complex on autophagosomes. Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation.sup.1,2. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes.sup.3,4,5,6,7. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex.sup.8,9,10,11, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion., Author(s): Jiajie Diao [sup.1] [sup.2] [sup.3] [sup.4] [sup.5] , Rong Liu [sup.6] [sup.7] [sup.8] , Yueguang Rong [sup.6] [sup.7] , Minglei Zhao [sup.1] [sup.2] [sup.3] [sup.4] [sup.5] , Jing Zhang [...]

The main organelles of the secretory and endocytic pathways--the endoplasmic reticulum (ER) and endosomes, respectively--are connected through contact sites whose numbers increase as endosomes mature (1-3.) One function of such sites is to enable dephosphorylation of the cytosolic tails of endosomal signalling receptors by an ER-associated phosphatase (4), whereas others serve to negatively control the association of endosomes with the minus-end-directed microtubule motor dynein (5) or mediate endosome fission (6). Cholesterol transfer and [Ca.sup.2+] exchange have been proposed as additional functions of such sites (2,3). However, the compositions, activities and regulations of ER-endosome contact sites remain incompletely understood. Here we show in human and rat cell lines that protrudin, an ER protein that promotes protrusion and neurite outgrowth (7), forms contact sites with late endosomes (LEs) via coincident detection of the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P). These contact sites mediate transfer of the microtubule motor kinesin 1 from protrudin to the motor adaptor FYCO1 on LEs. Repeated LE-ER contacts promote microtubule-dependent translocation of LEs to the cell periphery and subsequent synaptotagmin-VII-dependent fusion with the plasma membrane. Such fusion induces outgrowth of protrusions and neurites, which requires the abilities of protrudin and FYCO1 to interact with LEs and kinesin 1. Thus, protrudin-containing ER-LE contact sites are platforms for kinesin-1 loading onto LEs, and kinesin-1-mediated translocation of LEs to the plasma membrane, fuelled by repeated ER contacts, promotes protrusion and neurite outgrowth., The unusual presence of a PtdIns(3)P-binding FYVE domain in the ER protein protrudin (7-10) begged the question as to whether this protein interacts with endosomes, the major PtdIns(3)P-containing organelles in [...]

An emerging concept in the field of signal transduction is cAMP signal compartmentalization and localization in discrete locations of the cell and spatial localization dictating signaling specificity. In the pulmonary microvasculature, cAMP produced by membrane-bound adenylyl cyclase (AC) has been shown to be barrier protective, while cAMP produced by soluble AC is membrane disruptive. Several studies suggest that phosphodiesterases (PDEs) play a key role in regulating the spatial spread of cAMP signaling. PDE4 is primarily responsible for cAMP and PDE activity in pulmonary microvascular endothelial cells (PMVECs). However, the distribution of PDE4 isoforms in PMVECS and their contribution to cAMP compartmentalization is not well understood. We have begun utilizing hyperspectral FRET imaging and analysis techniques to determine the subcellular localization of cAMP signals in PMVECs. Wild-type and PDE4B knockout PMVECs were infected using a soluble Epac FRET probe (CFP-EPAC-YFP). We labeled nuclei (Hoechst), mitochondria (Mitotracker Red) and the plasma membrane (WGA-Tritc) for subcellular cAMP measurements. Time-lapse hyperspectral image data were acquired using an A1 spectral confocal microscope. A library containing the pure spectra of endmembers (CFP, YFP, Hoechst, Mitotracker red, Tritc) was used to determine the abundance of respective fluorophores/labels in the hyperspectral images. We then utilized automated and unbiased analysis techniques (Cell Profiler software), to measure the FRET efficiency in the subcellular compartments. We will assess cAMP levels using FRET efficiency that was measured in different subcellular compartments within the cell or across different cell types. In the future, we plan to assess the spatial distribution of PDE4 isoforms using isoform-specific antibodies and their role in maintaining endothelial permeability. The ability to measure cAMP levels in discrete subcellular compartments will also allow us to determine the role of different PDE4 isoforms in regulating compartmentalized cAMP signaling and thus in maintaining the endothelial barrier permeability. In addition to PDE4B KO cells, we will also use PDE4A and PDE4D KO cells to determine the role of each isoform in maintaining the endothelial permeability. Keywords: Hyperspectral Microscopy, FRET spectral Microscopy, Image Cytometry, Phosphodiesterases, cyclic AMP, and Subcellular second messenger signaling., INTRODUCTION An emerging concept in the field of signal transduction is that cAMP signals are compartmentalized and localized to discrete locations in the cell and that spatial localization dictates signaling [...]