scholarly journals Microtubule and Motor-Dependent Endocytic Vesicle Sorting in Vitro

2000 ◽  
Vol 151 (1) ◽  
pp. 179-186 ◽  
Author(s):  
Eustratios Bananis ◽  
John W. Murray ◽  
Richard J. Stockert ◽  
Peter Satir ◽  
Allan W. Wolkoff
Keyword(s):  
Essential Role ◽  
Immunofluorescence Microscopy ◽  
The Other ◽  
Endocytic Vesicle ◽  
In Vitro Reconstitution ◽  
Quantitative Immunofluorescence ◽  
Directed Motility ◽  
Endocytic Vesicles

Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor–ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by ∼300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end–directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5′-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 μM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.

Protein C Is Responsible for the Rapid Inactivation of Factor Va following Blood Clotting In Vitro

1994 ◽  
Vol 72 (01) ◽  
pp. 070-073
Author(s):  
Denise E Jackson ◽  
Christina A Mitchell ◽  
Hatem H Salem
Keyword(s):  
Protein C ◽  
Blood Clotting ◽  
Essential Role ◽  
The Other ◽  
Factor V ◽  
Factor Va ◽  
Rapid Inactivation ◽  
Protein C Activity

SummaryWhen whole blood is allowed to clot in vitro, factor V is rapidly activated to factor Va which is subsequently inactivated. We developed two monoclonal anti-protein C antibodies, one of which inhibits protein C activation and the other inhibits protein C activity. The addition of either antibody to blood before clotting in vitro significantly inhibited the inactivation of factor Va, confirming the essential role of protein C in mediating the rapid inactivation of factor Va.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

Cellular Samurai: katanin and the severing of microtubules

2000 ◽  
Vol 113 (16) ◽  
pp. 2821-2827 ◽  
Author(s):  
L. Quarmby
Keyword(s):  
Epithelial Cells ◽  
Essential Role ◽  
Aaa Atpase ◽  
C Elegans ◽  
Microtubule Severing ◽  
Biochemical Studies ◽  
New Evidence

Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

scholarly journals Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasunari Takami ◽  
Tatsuya Ono ◽  
Tatsuo Fukagawa ◽  
Kei-ichi Shibahara ◽  
Tatsuo Nakayama
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromatin Assembly ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

scholarly journals PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

1989 ◽  
Vol 9 (9) ◽  
pp. 3710-3719
Author(s):  
J Banroques ◽  
J N Abelson
Keyword(s):  
Essential Role ◽  
Mrna Splicing ◽  
Ribonucleoprotein Particle ◽  
Specific Antibodies ◽  
Small Nuclear Ribonucleoprotein ◽  
Assembly Pathway ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.

The Gastronomy of the Eye

2020 ◽  
pp. 175-198
Author(s):  
Vrushali Dhage
Keyword(s):  
Essential Role ◽  
Historical Perspective ◽  
Smart Cities ◽  
Methodological Approach ◽  
Economic Liberalization ◽  
The Other ◽  
Works Of Art

Works of art can be read at various levels: from being objects of simple retinal pleasure to the other extreme of being significant critical statements of their time. This chapter aims to strike a cerebral dialogue through the works of art. The current study shall consider the latter function of art and analyze the methods in which contemporary Indian artists have made attempts to provide a critique of the early initiatives towards developing Delhi and Mumbai as ‘smart cities'. The review of works from India concludes the essential role of infrastructural projects and envisioned spaces built in the era of economic liberalization. The study aims at drawing a methodological approach, with an art historical perspective, with the artists analysing and translating the urban experiential phenomenon, into artworks.

THE ROLE OF ADSORPTION IN CONTROLLING THE RATE OF REACTION OF CHLORAMBUCIL WITH PROTEIN

1963 ◽  
Vol 41 (4) ◽  
pp. 931-939 ◽  
Author(s):  
J. H. Linford
Keyword(s):  
Molecular Weight ◽  
Carboxylic Acid ◽  
Bovine Serum Albumin ◽  
Inverse Relationship ◽  
Reaction Rate ◽  
The Other ◽  
Alkylation Reaction ◽  
Rate Of Reaction

Two proteins, haemoglobin and bovine serum albumin, have been studied with respect to their rates of alkylation by chlorambucil in vitro at 37 °C and pH 8.4. The proteins are of nearly the same molecular weight and free carboxylic acid content, but the alkylation reaction is 30 times faster with haemoglobin. On the other hand, the adsorption of chlorambucil by albumin is 20 times greater than that exhibited by haemoglobin. This inverse relationship between extent of adsorption and reaction rate suggests that adsorption protects the chlorambucil from activation in the solvent.

scholarly journals RNA polymerase II plays an active role in the formation of gene loops through the Rpb4 subunit

2019 ◽  
Vol 47 (17) ◽  
pp. 8975-8987 ◽  
Author(s):  
Paula Allepuz-Fuster ◽  
Michael J O’Brien ◽  
Noelia González-Polo ◽  
Bianca Pereira ◽  
Zuzer Dhoondia ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Essential Role ◽  
Transcription Termination ◽  
Active Role ◽  
The Other ◽  
Loop Formation ◽  
Direct Role

AbstractGene loops are formed by the interaction of initiation and termination factors occupying the distal ends of a gene during transcription. RNAPII is believed to affect gene looping indirectly owing to its essential role in transcription. The results presented here, however, demonstrate a direct role of RNAPII in gene looping through the Rpb4 subunit. 3C analysis revealed that gene looping is abolished in the rpb4Δ mutant. In contrast to the other looping-defective mutants, rpb4Δ cells do not exhibit a transcription termination defect. RPB4 overexpression, however, rescued the transcription termination and gene looping defect of sua7-1, a mutant of TFIIB. Furthermore, RPB4 overexpression rescued the ssu72-2 gene looping defect, while SSU72 overexpression restored the formation of gene loops in rpb4Δ cells. Interestingly, the interaction of TFIIB with Ssu72 is compromised in rpb4Δ cells. These results suggest that the TFIIB–Ssu72 interaction, which is critical for gene loop formation, is facilitated by Rpb4. We propose that Rpb4 is promoting the transfer of RNAPII from the terminator to the promoter for reinitiation of transcription through TFIIB–Ssu72 mediated gene looping.

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