Membrane detachment is not essential for COG complex function.
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ABSTRACT:
SUBMITTER: Climer LK
PROVIDER: S-EPMC5896934 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Multisubunit tethering complexes of the CATCHR (complexes associated with tethering containing helical rods) family are proposed to mediate the initial contact between an intracellular trafficking vesicle and its membrane target. To begin elucidating the molecular architecture of one well-studied example, the conserved oligomeric Golgi (COG) complex, we reconstituted its essential subunits (Cog1, Cog2, Cog3 and Cog4) and used single-particle electron microscopy to reveal a y-shaped structure with three flexible, highly extended legs. Labeling experiments established that the N termini of all four subunits interact along the proximal segment of one leg, whereas three of the four C termini are located at the tips of the legs. Our results suggest that the central region of the Cog1-Cog2-Cog3-Cog4 complex, as well as the distal regions of at least two legs, all participate in interactions with other components of the intracellular trafficking machinery.
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Project description:The conserved oligomeric Golgi (COG) complex orchestrates vesicular trafficking to and within the Golgi apparatus. Here, we use negative-stain electron microscopy to elucidate the architecture of the hetero-octameric COG complex from Saccharomyces cerevisiae. Intact COG has an intricate shape, with four (or possibly five) flexible legs, that differs strikingly from that of the exocyst complex and appears to be well suited for vesicle capture and fusion.
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Project description:RationaleJunctional membrane complexes (JMCs) in myocytes are critical microdomains, in which excitation-contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. However, the role of newly identified JMC protein SPEG (striated muscle preferentially expressed protein kinase) remains unclear.ObjectiveTo determine the role of SPEG in healthy and failing adult hearts.Methods and resultsProteomic analysis of immunoprecipitated JMC proteins ryanodine receptor type 2 and junctophilin-2 (JPH2) followed by mass spectrometry identified the serine-threonine kinase SPEG as the only novel binding partner for both proteins. Real-time polymerase chain reaction revealed the downregulation of SPEG mRNA levels in failing human hearts. A novel cardiac myocyte-specific Speg conditional knockout (MCM-Spegfl/fl) model revealed that adult-onset SPEG deficiency results in heart failure (HF). Calcium (Ca2+) and transverse-tubule imaging of ventricular myocytes from MCM-Spegfl/fl mice post HF revealed both increased sarcoplasmic reticulum Ca2+ spark frequency and disrupted JMC integrity. Additional studies revealed that transverse-tubule disruption precedes the development of HF development in MCM-Spegfl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM-Spegfl/fl mice, suggesting that loss of SPEG phosphorylation of JPH2 led to transverse-tubule disruption, a precursor of HF development in SPEG-deficient mice.ConclusionsThe novel JMC protein SPEG is downregulated in human failing hearts. Acute loss of SPEG in mouse hearts causes JPH2 dephosphorylation and transverse-tubule loss associated with downstream Ca2+ mishandling leading to HF. Our study suggests that SPEG could be a novel target for the treatment of HF.
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Project description:Of the four separate PE biosynthetic pathways in eukaryotes, one occurs in the mitochondrial inner membrane (IM) and is executed by phosphatidylserine decarboxylase (Psd1). Deletion of Psd1 is lethal in mice and compromises mitochondrial function. We hypothesize that this reflects inefficient import of non-mitochondrial PE into the IM. Here, we test this by re-wiring PE metabolism in yeast by re-directing Psd1 to the outer mitochondrial membrane or the endomembrane system and show that PE can cross the IMS in both directions. Nonetheless, PE synthesis in the IM is critical for cytochrome bc1 complex (III) function and mutations predicted to disrupt a conserved PE-binding site in the complex III subunit, Qcr7, impair complex III activity similar to PSD1 deletion. Collectively, these data challenge the current dogma of PE trafficking and demonstrate that PE made in the IM by Psd1 support the intrinsic functionality of complex III.
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Project description:Spatially and temporally regulated membrane trafficking events incorporate membrane and cell wall materials into the pollen tube apex and are believed to underlie the rapid pollen tube growth. In plants, the molecular mechanisms and physiological functions of intra-Golgi transport and Golgi integrity maintenance remain largely unclear. The conserved oligomeric Golgi (COG) complex has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells. Using genetic and cytologic approaches, we demonstrate that T-DNA insertions in Arabidopsis COG complex subunits, COG3 and COG8, cause an absolute, male-specific transmission defect that can be complemented by expression of COG3 and COG8 from the LAT52 pollen promoter, respectively. No obvious abnormalities in the microgametogenesis of the two mutants are observed, but in vitro and in vivo pollen tube growth are defective. COG3 or COG8 proteins fused to green fluorescent protein (GFP) label the Golgi apparatus. In pollen of both mutants, Golgi bodies exhibit altered morphology. Moreover, ?-COP and EMP12 proteins lose their tight association with the Golgi. These defects lead to the incorrect deposition of cell wall components and proteins during pollen tube growth. COG3 and COG8 interact directly with each other, and a structural model of the Arabidopsis COG complex is proposed. We believe that the COG complex helps to modulate Golgi morphology and vesicle trafficking homeostasis during pollen tube tip growth.
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Project description:Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. "Deep etch" EM of purified COG revealed an approximately 37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.
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Project description:The Golgi apparatus is a central station for protein trafficking in eukaryotic cells. A widely accepted model of protein transport within the Golgi apparatus is cisternal maturation. Each cisterna has specific resident proteins, which are thought to be maintained by COPI-mediated transport. However, the mechanisms underlying specific sorting of these Golgi-resident proteins remain elusive. To obtain a clue to understand the selective sorting of vesicles between the Golgi cisterenae, we investigated the molecular arrangements of the conserved oligomeric Golgi (COG) subunits in yeast cells. Mutations in COG subunits cause defects in Golgi trafficking and glycosylation of proteins and are causative of Congenital Disorders of Glycosylation (CDG) in humans. Interactions among COG subunits in cytosolic and membrane fractions were investigated by co-immunoprecipitation. Cytosolic COG subunits existed as octamers, whereas membrane-associated COG subunits formed a variety of subcomplexes. Relocation of individual COG subunits to mitochondria resulted in recruitment of only a limited number of other COG subunits to mitochondria. These results indicate that COG proteins function in the forms of a variety of subcomplexes and suggest that the COG complex does not comprise stable tethering without other interactors.Key words: The Golgi apparatus, COG complex, yeast, membrane trafficking, multi-subunit tethering complex.
| S-EPMC6116515 | biostudies-literature
Project description:The conserved oligomeric Golgi complex is a peripheral membrane protein complex that orchestrates the tethering and fusion of intra-Golgi transport carriers with Golgi membranes. In this study we have investigated the membrane attachment of the COG complex and it's on/off dynamic on Golgi membranes. Several complimentary approaches including knock-sideways depletion, FRAP, and FLIP revealed that assembled COG complex is not diffusing from Golgi periphery in live HeLa cells. Moreover, COG subunits remained membrane-associated even in COG4 and COG7 depleted cells when Golgi architecture was severely affected. Overexpression of myc-tagged COG sub-complexes revealed that different membrane-associated COG partners including β-COP, p115 and SNARE STX5 preferentially bind to different COG assemblies, indicating that COG subunits interact with Golgi membranes in a multipronged fashion.
| S-EPMC3948154 | biostudies-literature
Project description:The force-induced detachment of the adhesion protein complex CD2-CD58 was studied by steered molecular dynamics simulations. The forced detachment of CD2 and CD58 shows that the system can respond to an external force by two mechanisms, which depend on the loading rate. At the rapid loading rates of 70 and 35 pN/ps (pulling speeds of 1 and 0.5 A/ps) the two proteins unfold before they separate, whereas at slower loading rates of 7 and 3.5 pN/ps (pulling speeds of 0.1 and 0.05 A/ps), the proteins separate before the domains can unfold. When subjected to a constant force of 400 pN, the two proteins separated without significant structural distortion. These findings suggest that protein unfolding is not coupled to the adhesive function of CD2 and CD58. The simulations further confirm that salt bridges primarily determine the tensile strength of the protein-to-protein bond, and that the order of salt bridge rupture depends mainly on the position of the bond, relative to the line of action of the applied force. Salt bridges close to this line break first. The importance of each of the salt bridges for adhesion, determined from the simulations, correlates closely with their role in cell-to-cell adhesion and equilibrium binding determined by site-directed mutagenesis experiments.
| S-EPMC1302789 | biostudies-literature
Project description:BackgroundThe Conserved Oligomeric Golgi (COG) complex is an eight-subunit assembly that localizes peripherally to Golgi membranes and is involved in retrograde vesicular trafficking. COG subunits are organized in two heterotrimeric groups, Cog2, -3, -4 and Cog5, -6, -7, linked by a dimeric group formed by Cog1 and Cog8. Dysfunction of COG complex in humans has been associated with new forms of Congenital Disorders of Glycosylation (CDG), therefore highlighting its essential role. In the present study, we intended to gain further insights into the evolution of COG subunits in vertebrates, using comparative analyses of all eight COG proteins.ResultsWe used protein distances and dN/dS ratios as a measure of the rate of proteins evolution. The results showed that all COG subunits are evolving under strong purifying selection, although COG1 seems to evolve faster than the remaining proteins. In addition, we also tested the expression of COG genes in 20 human tissues, and demonstrate their ubiquitous nature.ConclusionsCOG complex has a critical role in Golgi structure and function, which, in turn, is involved in protein sorting and glycosylation. The results of this study suggest that COG subunits are evolutionary constrained to maintain the interactions between each other, as well with other partners involved in vesicular trafficking, in order to preserve both the integrity and function of the complex.
| S-EPMC2927914 | biostudies-literature