scholarly journals PtdIns4P recognition by Vps74/GOLPH3 links PtdIns 4-kinase signaling to retrograde Golgi trafficking

2009 ◽  
Vol 187 (7) ◽  
pp. 967-975 ◽  
Author(s):  
Christopher S. Wood ◽  
Karl R. Schmitz ◽  
Nicholas J. Bessman ◽  
Thanuja Gangi Setty ◽  
Kathryn M. Ferguson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mitochondrial Dna ◽  
Golgi Apparatus ◽  
Integral Membrane Proteins ◽  
Golgi Membrane ◽  
Sulfate Ion ◽  
Sensitive Factor ◽  
Cytosolic Domains

Targeting and retention of resident integral membrane proteins of the Golgi apparatus underly the function of the Golgi in glycoprotein and glycolipid processing and sorting. In yeast, steady-state Golgi localization of multiple mannosyltransferases requires recognition of their cytosolic domains by the peripheral Golgi membrane protein Vps74, an orthologue of human GOLPH3/GPP34/GMx33/MIDAS (mitochondrial DNA absence sensitive factor). We show that targeting of Vps74 and GOLPH3 to the Golgi apparatus requires ongoing synthesis of phosphatidylinositol (PtdIns) 4-phosphate (PtdIns4P) by the Pik1 PtdIns 4-kinase and that modulation of the levels and cellular location of PtdIns4P leads to mislocalization of these proteins. Vps74 and GOLPH3 bind specifically to PtdIns4P, and a sulfate ion in a crystal structure of GOLPH3 indicates a possible phosphoinositide-binding site that is conserved in Vps74. Alterations in this site abolish phosphoinositide binding in vitro and Vps74 function in vivo. These results implicate Pik1 signaling in retention of Golgi-resident proteins via Vps74 and show that GOLPH3 family proteins are effectors of Golgi PtdIns 4-kinases.

Insights into the Secretory Pathway, the Mechanism of Terminal Glycosylation and Intracellular Peptide Hormone Receptors from Studies on Hepatic Golgi Fractions

1981 ◽  
Vol 39 ◽  
pp. 516-519
Author(s):  
J.J.M. Bergeron ◽  
B.I. Posner ◽  
Jacques Paiement ◽  
R. Sikstrom ◽  
M. Khan
Keyword(s):  
Golgi Apparatus ◽  
Plasma Proteins ◽  
Secretory Pathway ◽  
Hormone Receptors ◽  
Peptide Hormone ◽  
Secretory Protein ◽  
Golgi Membrane ◽  
Membrane Structures

Recent studies on purified subcellular fractions of hepatic Golgi apparatus have provided insight into the functioning of the Golgi apparatus in vivo.The hepatocyte is the site of synthesis of most circulating plasma proteins. On a total protein basis, purified Golgi fractions revealed mainly secretory content (albumin, transferrin and other plasma proteins) as major constituents. After an in vivo injection of radiolabeled leucine, newly synthesized secretory protein followed a temporal route from cis to trans regions of Golgi apparatus before appearance in the plasma. This route was revealed by studies on disrupted Golgi fractions enriched in disparate regions of the Golgi apparatus.The terminal glycosylation of secretory glcyoproteins (e.g. transferrin) can be studied by observing the transfer of UDP-(3H)-galactose to endogenous acceptors within Golgi fractions. Transfer was shown to occur to a glycolipid (dolichyl galactosyl phosphate) probably on the cytosolic aspect of the Golgi membrane. Translocation of the labeled galactose across the membrane coincided with fusion of Golgi saccules in vitro. It is felt that during the process of Golgi membrane fusion, inverted lipid- micellar membrane structures translocate the dolichyl galactosyl phosphate from a cytosolic to a luminal orientation. Luminally oriented dolichyl galactosyl phosphate would then serve as substrate for galactose transfer to intraluminal glycopeptide acceptors via intraluminal galactosyl transferase enzyme.

scholarly journals Group II intron domain 5 facilitates a trans-splicing reaction.

1988 ◽  
Vol 8 (6) ◽  
pp. 2361-2366 ◽  
Author(s):  
K A Jarrell ◽  
R C Dietrich ◽  
P S Perlman
Keyword(s):  
Mitochondrial Dna ◽  
Group Ii Intron ◽  
Intron Sequence ◽  
Trans Splicing ◽  
Group Ii ◽  
Self Splicing ◽  
Yeast Mitochondrial Dna ◽  
Domain 4

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.

Effects of dietary alteration of bicarbonate and magnesium on rat bone

1986 ◽  
Vol 250 (2) ◽  
pp. F302-F307 ◽  
Author(s):  
J. M. Burnell ◽  
C. Liu ◽  
A. G. Miller ◽  
E. Teubner
Keyword(s):  
Crystal Structure ◽  
Bone Formation ◽  
X Ray Diffraction ◽  
X Ray ◽  
Growing Rats ◽  
Final Composition ◽  
Rat Bone

To study the effects of bicarbonate and magnesium on bone, mild acidosis and/or hypermagnesemia were produced in growing rats by feeding ammonium chloride and/or magnesium sulfate. Bone composition, quantitative histomorphometry, and mineral x-ray diffraction (XRD) characteristics were measured after 6 wk of treatment. The results demonstrated that both acidosis (decreased HCO3) and hypermagnesemia inhibited periosteal bone formation, and, when combined, results were summative; and the previously observed in vitro role of HCO3- and Mg2+ as inhibitors of crystal growth were confirmed in vivo. XRD measurements demonstrated that decreased plasma HCO3 resulted in larger crystals and increased Mg resulted in smaller crystals. However, the combined XRD effects of acidosis and hypermagnesemia resembled acidosis alone. It is postulated that the final composition and crystal structure of bone are strongly influenced by HCO3- and Mg2+, and the effects are mediated by the combined influence on both osteoblastic bone formation and the growth of hydroxyapatite.

DNA Synthesis in Isolated Yeast Mitochondria

1974 ◽  
Vol 52 (11) ◽  
pp. 941-949 ◽  
Author(s):  
L. Zeman ◽  
C. V. Lusena
Keyword(s):  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Base Composition ◽  
Nuclear Dna ◽  
Sedimentation Velocity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Denatured State ◽  
Saccharomyces Cerevisiae Mitochondria

Isolated yeast (Saccharomyces cerevisiae) mitochondria incorporate radioactive precursors into mitochondrial DNA. This in vitro labelled DNA was characterized by isopycnic and sedimentation velocity centrifugation both in the native and denatured state. The profiles of isopycnic CsCl gradients obtained by centrifugation in a fixed-angle rotor are skewed toward high density. The skew is neither due to the presence of in vitro labelled nuclear DNA nor due to random breaks in mitochondrial DNA which would reveal, then, its heterogeneity in base composition. The in vitro labelled DNA is reproducibly recovered as a class of molecules sedimenting at about 5–8 S, indicating a molecular weight of 1 × 105 – 4 × 105 daltons, while the smallest in vivo labelled fragments sediment at about 13–14 S, corresponding to 1.6 × 106 – 2.0 × 106 daltons. After denaturation, the in vitro labelled DNA molecules sediment at about 2–5 S, corresponding to a single-strand molecular weight of 1 × 104 – 7 × 104 daltons, which is about one hundred times less than the observed size of the denatured in vivo labelled molecules.

Ultrastructural analysis of Drosophila ovarian follicles differing in yolk polypeptide (yps) composition

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 319-328
Author(s):  
F. Giorgi ◽  
P. Lucchesi ◽  
A. Morelli ◽  
M. Bownes
Keyword(s):  
Golgi Apparatus ◽  
Juvenile Hormone ◽  
Developmental Stages ◽  
Ovarian Follicles ◽  
Endocytic Pathway ◽  
Intermediate Cell ◽  
Wild Type ◽  
Vesicular Traffic

Drosophila ovarian follicles were examined ultrastructurally to study the vesicular traffic in the cortical ooplasm. The endocytic pathway leading to the production of yolk spheres was visualized following in vivo or in vitro exposure to peroxidase. The Golgi apparatus and the yolk spheres of wild-type ovarian follicles were preferentially labelled by fixation with osmium zinc iodide (OZI). Labelling of wild-type ovarian follicles was compared to that of several mutant follicles--L186/Basc, fs(2)A17 and ap4--which are defective in vitellogenesis. In these mutants, the Golgi apparatus and the vesicles nearby were either scantly labelled or not labelled at all. In oocytes from flies homozygous for the gene fs(1)1163, the Golgi apparatus was labelled as in the controls, but no yolk spheres appeared to be labelled with OZI at any of the developmental stages. In several Drosophila strains, the pattern of OZI label in the cortical ooplasm was seen to vary in relation to the number of yp structural genes. In starved Drosophila females, OZI labelling of the cortical ooplasm appeared restricted to the Golgi apparatus and to an extended tubular network. A similar labelling pattern was also detected in in vitro cultured vitellogenic follicles. Refeeding, topical application of juvenile hormone analogue to starved females or hormone addition to the culture medium, all caused the yolk spheres to become labelled with OZI and to incorporate peroxidase. These observations prove that impairing endocytic uptake by either mutation or lack of juvenile hormone prevents fusion of coated vesicles and tubules with the yolk spheres and leads them instead to form an intermediate cell compartment with Golgi-derived vesicles.

scholarly journals Comparative analysis of different nuclear transfer techniques to prevent the transmission of mitochondrial DNA variants

2019 ◽  
Author(s):  
M Tang ◽  
R R Guggilla ◽  
Y Gansemans ◽  
M Van der Jeught ◽  
A Boel ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Disease Transmission ◽  
Nuclear Transfer ◽  
Smooth Endoplasmic Reticulum ◽  
Scientific Evidence ◽  
Polar Body ◽  
Comparable Rate ◽  
Carry Over

Abstract Prevention of mitochondrial DNA (mtDNA) diseases may currently be possible using germline nuclear transfer (NT). However, scientific evidence to compare efficiency of different NT techniques to overcome mtDNA diseases is lacking. Here, we performed four types of NT, including first or second polar body transfer (PB1/2T), maternal spindle transfer (ST) and pronuclear transfer (PNT), using NZB/OlaHsd and B6D2F1 mouse models. Embryo development was assessed following NT and mtDNA carry-over levels were measured by next generation sequencing (NGS). Moreover, we explored two novel protocols (PB2T-a and PB2T-b) to optimize PB2T using mouse and human oocytes. Chromosomal profiles of NT-generated blastocysts were evaluated using NGS. In mouse, our findings reveal that only PB2T-b successfully leads to blastocysts. There were comparable blastocyst rates amongst PB1T, PB2T-b, ST and PNT embryos. Furthermore, PB1T and PB2T-b had lower mtDNA carry-over levels than ST and PNT. After extrapolation of novel PB2T-b to human in vitro matured (IVM) oocytes and in vivo matured oocytes with smooth endoplasmic reticulum aggregates (SERa) oocytes, the reconstituted embryos successfully developed to blastocysts at a comparable rate to ICSI controls. PB2T-b embryos generated from IVM oocytes showed a similar euploidy rate to ICSI controls. Nevertheless, our mouse model with non-mutated mtDNAs is different from a mixture of pathogenic and non-pathogenic mtDNAs in a human scenario. Novel PB2T-b requires further optimization to improve blastocyst rates in human. Although more work is required to elucidate efficiency and safety of NT, our study suggests that PBT may have the potential to prevent mtDNA disease transmission.

scholarly journals Oxidative damage to mitochondrial DNA and glutathione oxidation in apoptosis: studies in vivo and in vitro

1999 ◽  
Vol 13 (9) ◽  
pp. 1055-1064 ◽  
Author(s):  
J. M. Esteve ◽  
J. Mompo ◽  
J. Garcia Asuncion ◽  
J. Sastre ◽  
M. Asensi ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Oxidative Damage ◽  
Glutathione Oxidation

scholarly journals Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo

2007 ◽  
Vol 178 (3) ◽  
pp. 465-476 ◽  
Author(s):  
Insa Geffers ◽  
Katrin Serth ◽  
Gavin Chapman ◽  
Robert Jaekel ◽  
Karin Schuster-Gossler ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Mouse Embryos ◽  
Functional Equivalence ◽  
Presomitic Mesoderm ◽  
Drosophila Wing ◽  
Wing Discs ◽  
Golgi Network ◽  
Notch Ligands

The Notch ligands Dll1 and Dll3 are coexpressed in the presomitic mesoderm of mouse embryos. Despite their coexpression, mutations in Dll1 and Dll3 cause strikingly different defects. To determine if there is any functional equivalence, we replaced Dll1 with Dll3 in mice. Dll3 does not compensate for Dll1; DLL1 activates Notch in Drosophila wing discs, but DLL3 does not. We do not observe evidence for antagonism between DLL1 and DLL3, or repression of Notch activity in mice or Drosophila. In vitro analyses show that differences in various domains of DLL1 and DLL3 individually contribute to their biochemical nonequivalence. In contrast to endogenous DLL1 located on the surface of presomitic mesoderm cells, we find endogenous DLL3 predominantly in the Golgi apparatus. Our data demonstrate distinct in vivo functions for DLL1 and DLL3. They suggest that DLL3 does not antagonize DLL1 in the presomitic mesoderm and warrant further analyses of potential physiological functions of DLL3 in the Golgi network.

199 EVIDENCE FOR A NOVEL PERTURBATION IN CLONED FETUSES: MITOCHONDRIAL DNA DEPLETION

2007 ◽  
Vol 19 (1) ◽  
pp. 216
Author(s):  
S. Hiendleder ◽  
D. Bebbere ◽  
S. E. Ulbrich ◽  
V. Zakhartchenko ◽  
M. Weppert ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Nuclear Gene ◽  
Complement C3 ◽  
Mtdna Copy Number ◽  
Vitro Fertilization ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Dna Depletion

The reported mtDNA turnover and plasticity of mtDNA copy number in mammalian zygotes and early embryos (McConnel and Petrie 2004 Reprod. Biomed. Online 9, 418–424) have revealed a potential for adverse effects of in vitro embryo techniques on mtDNA and mitochondrial function. We explored the effects of in vitro fertilization (IVF) and somatic cell nuclear transfer cloning (NT) on relative mtDNA amount and phenotype in viable bovine fetuses recovered 80 days after the initiation of embryonic development (Hiendleder et al. 2004 Biol. Reprod. 71, 217–223). We sampled brain, liver, and skeletal muscle to represent all 3 embryonic germ layers, and compared IVF-fetuses (n = 24), NT-fetuses (n = 23), and fetuses generated by in vivo insemination (controls, n = 24). This experimental approach allowed us to distinguish abnormalities specific to cloning from more general consequences of in vitro embryo manipulation. We analyzed relative mtDNA amounts by real-time quantitative PCR (qPCR) and amplified a segment of the mtDNA control region that was normalized against the nuclear gene complement C3. ANOVA (SPSS 13.0) of qPCR data and phenotypic parameters revealed significant effects of fetus group on mtDNA amount in liver (P < 0.05) and muscle (P < 0.01), and on fetus (P < 0.001), heart (P < 0.001), and liver (P < 0.001) weights. The mtDNA amount in all tissues from IVF-fetuses was normal, but mtDNA levels in liver (-23%; P < 0.05) and muscle (-24%; P < 0.01) of NT-fetuses were significantly lower than in controls. Fetuses derived from IVF- or NT-embryos were similar in weight and displayed fetal overgrowth (+19% and +22%; P < 0.001), but only the NT-fetuses were affected by disproportionate hepatomegaly and cardiomegaly with 31% and 49% increases (ANCOVA; P < 0.001) in their respective organ weights. This further partitioned NT-fetuses from IVF-fetuses and identified symptoms that are also encountered in mitochondrial DNA depletion syndromes (MDDS): a phenotypically heterogeneous group of human disorders characterized by loss of mtDNA from various tissues during development and associated respiratory chain dysfunction. The MDDS phenotypes have mainly been classified into a hepatocerebral (MIM 251880) or myopathic (MIM 609560) form, and neonates and infants display a spectrum of abnormalities, including hepatomegaly and cardiomegaly, that are similar or identical to phenotypic abnormalities commonly encountered in cloned mammals. Reduced mtDNA amounts in NT-fetuses could stem from perturbation of mtDNA during the reported turnover period, or be a secondary effect of epigenetic change in nuclear-encoded genes involved in mtDNA replication and stability. Mitochondrial transcription factor A (TFAM) is regulated by CpG methylation in vitro, but our real-time RT-PCR quantification of TFAM transcript in liver and muscle of a subset of NT- and control fetuses failed to detect significant differences (P > 0.10). In conclusion, our observed reduction of mtDNA amount in cloned fetuses provides the molecular basis for a mitochondrial perspective on pathological phenotypes of cloned mammals, and may explain similarities to mitochondrial disease in human.

scholarly journals Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 872 ◽  
Author(s):  
Clemens Grimm ◽  
Jann-Patrick Pelz ◽  
Cornelius Schneider ◽  
Katrin Schäffler ◽  
Utz Fischer
Keyword(s):  
Crystal Structure ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Turnover Rates ◽  
Mrna Transcription ◽  
Terminal Part ◽  
Protein Output

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.

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