scholarly journals Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation

2021 ◽  
Author(s):  
Sudeep Banjade ◽  
Lu Zhu ◽  
Jeffrey Jorgensen ◽  
Sho Suzuki ◽  
Scott D. Emr
Keyword(s):  
Membrane Proteins ◽  
Self Assembly ◽  
Cell Biology ◽  
Substrate Recognition ◽  
Initial Step ◽  
Binding Interactions ◽  
Vacuole Membrane ◽  
Multivalent Interactions ◽  
Cargo Sorting

AbstractThe general mechanisms by which ESCRTs are specifically recruited to various membranes, and how ESCRT subunits are spatially organized remain central questions in cell biology. At the endosome and lysosomes, ubiquitination of membrane proteins triggers ESCRT-mediated substrate recognition and degradation. Using the yeast lysosome/vacuole, we define the principles by which substrate engagement by ESCRTs occurs at this organelle. We find that multivalent interactions between ESCRT-0 and polyubiquitin is critical for substrate recognition at yeast vacuoles, with a lower-valency requirement for cargo engagement at endosomes. Direct recruitment of ESCRT-0 induces dynamic foci on the vacuole membrane, and forms fluid condensates in vitro with polyubiquitin. We propose that self-assembly of early ESCRTs induces condensation, an initial step in ESCRT-assembly/nucleation at membranes. This property can be tuned specifically at various organelles by modulating the number of binding interactions.One-Sentence SummaryCondensation of multivalent ESCRT-0/polyubiquitin assemblies organizes cargo sorting reactions at lysosomes

scholarly journals Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

2019 ◽  
Vol 20 (14) ◽  
pp. 3479 ◽  
Author(s):  
Jean-Denis Pedelacq ◽  
Stéphanie Cabantous
Keyword(s):  
Protein Interactions ◽  
Self Assembly ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein Detection ◽  
Molecular Engineering ◽  
Fluorescent Markers ◽  
Diverse Area ◽  
Green Fluorescent

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.

scholarly journals One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

2015 ◽  
Vol 113 (3) ◽  
pp. 608-613 ◽  
Author(s):  
Peter J. Yunker ◽  
Haruichi Asahara ◽  
Kuo-Chan Hung ◽  
Corey Landry ◽  
Laura R. Arriaga ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Membrane Proteins ◽  
Self Assembly ◽  
Hydrophobic Interactions ◽  
Cell Communication ◽  
Transmembrane Helices ◽  
One Pot ◽  
Surface Receptors ◽  
Coupling Protein

Single-span membrane proteins (ssMPs) represent approximately one-half of all membrane proteins and play important roles in cellular communications. However, like all membrane proteins, ssMPs are prone to misfolding and aggregation because of the hydrophobicity of transmembrane helices, making them difficult to study using common aqueous solution-based approaches. Detergents and membrane mimetics can solubilize membrane proteins but do not always result in proper folding and functionality. Here, we use cell-free protein synthesis in the presence of oil drops to create a one-pot system for the synthesis, assembly, and display of functional ssMPs. Our studies suggest that oil drops prevent aggregation of some in vitro-synthesized ssMPs by allowing these ssMPs to localize on oil surfaces. We speculate that oil drops may provide a hydrophobic interior for cotranslational insertion of the transmembrane helices and a fluidic surface for proper assembly and display of the ectodomains. These functionalized oil drop surfaces could mimic cell surfaces and allow ssMPs to interact with cell surface receptors under an environment closest to cell–cell communication. Using this approach, we showed that apoptosis-inducing human transmembrane proteins, FasL and TRAIL, synthesized and displayed on oil drops induce apoptosis of cultured tumor cells. In addition, we take advantage of hydrophobic interactions of transmembrane helices to manipulate the assembly of ssMPs and create artificial clusters on oil drop surfaces. Thus, by coupling protein synthesis with self-assembly at the water–oil interface, we create a platform that can use recombinant ssMPs to communicate with cells.

scholarly journals ESCRTs function directly on the lysosome membrane to downregulate ubiquitinated lysosomal membrane proteins

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lu Zhu ◽  
Jeff R Jorgensen ◽  
Ming Li ◽  
Ya-Shan Chuang ◽  
Scott D Emr
Keyword(s):  
Membrane Proteins ◽  
Underlying Mechanism ◽  
Genetic Screens ◽  
Vacuole Membrane ◽  
Cargo Delivery ◽  
Ubiquitin Ligase Complex ◽  
Lysosomal Membrane Proteins ◽  
Homeostasis Regulation ◽  
Nutrient Homeostasis ◽  
Cargo Sorting

The lysosome plays an important role in maintaining cellular nutrient homeostasis. Regulation of nutrient storage can occur by the ubiquitination of certain transporters that are then sorted into the lysosome lumen for degradation. To better understand the underlying mechanism of this process, we performed genetic screens to identify components of the sorting machinery required for vacuole membrane protein degradation. These screens uncovered genes that encode a ubiquitin ligase complex, components of the PtdIns 3-kinase complex, and the ESCRT machinery. We developed a novel ubiquitination system, Rapamycin-Induced Degradation (RapiDeg), to test the sorting defects caused by these mutants. These tests revealed that ubiquitinated vacuole membrane proteins recruit ESCRTs to the vacuole surface, where they mediate cargo sorting and direct cargo delivery into the vacuole lumen. Our findings demonstrate that the ESCRTs can function at both the late endosome and the vacuole membrane to mediate cargo sorting and intra-luminal vesicle formation.

scholarly journals Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteins in vitro

2000 ◽  
Vol 97 (4) ◽  
pp. 1473-1476 ◽  
Author(s):  
I. Simidjiev ◽  
S. Stoylova ◽  
H. Amenitsch ◽  
T. Javorfi ◽  
L. Mustardy ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Self Assembly ◽  
Integral Membrane Proteins

Can we identify the forces that drive the folding of integral membrane proteins?

2001 ◽  
Vol 29 (4) ◽  
pp. 408-413 ◽  
Author(s):  
P. J. Booth ◽  
R. H. Templer ◽  
A. R. Curran ◽  
S. J. Allen
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Cell Biology ◽  
Large Body ◽  
Integral Membrane Proteins ◽  
Generic Properties ◽  
Membrane Bilayer ◽  
Rate Limiting

Protein folding has been at the forefront of molecular cell biology research for several years. However, integral membrane proteins have eluded detailed molecular level study until recently. One reason is the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is nevertheless a large body of information on lipid properties and in particular on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to design efficient in vitro, lipid-bilayer folding systems for membrane proteins. Bacteriorhodopsin has been used as a model system for our initial studies: we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency seem to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer and the lateral pressure that the lipid chains exert on their neighbouring folding protein. These are generic properties of the bilayer that can be achieved with simple mixtures of many types of biological lipid and seem to be important in vivo.

scholarly journals A Comprehensive Tutorial onIn VitroCharacterization of New Photosensitizers for Photodynamic Antitumor Therapy and Photodynamic Inactivation of Microorganisms

2013 ◽  
Vol 2013 ◽  
pp. 1-17 ◽  
Author(s):  
Tobias Kiesslich ◽  
Anita Gollmer ◽  
Tim Maisch ◽  
Mark Berneburg ◽  
Kristjan Plaetzer
Keyword(s):  
Photodynamic Therapy ◽  
Cell Biology ◽  
Wide Spectrum ◽  
Photophysical Properties ◽  
Intracellular Localization ◽  
Initial Step ◽  
Photodynamic Inactivation ◽  
Comprehensive Collection ◽  
Light Protection

In vitroresearch performed on eukaryotic or prokaryotic cell cultures usually represents the initial step for characterization of a novel photosensitizer (PS) intended for application in photodynamic therapy (PDT) of cancer or photodynamic inactivation (PDI) of microorganisms. Although many experimental steps of PS testing make use of the wide spectrum of methods readily employed in cell biology, special aspects of working with photoactive substances, such as the autofluorescence of the PS molecule or the requirement of light protection, need to be considered when performingin vitroexperiments in PDT/PDI. This tutorial represents a comprehensive collection of operative instructions, by which, based on photochemical and photophysical properties of a PS, its uptake into cells, the intracellular localization and photodynamic action in both tumor cells and microorganisms novel photoactive molecules may be characterized for their suitability for PDT/PDI. Furthermore, it shall stimulate the efforts to expand the convincing benefits of photodynamic therapy and photodynamic inactivation within both established and new fields of applications and motivate scientists of all disciplines to get involved in photodynamic research.

scholarly journals Synthesis of Extended, Self-Assembled Biological Membranes containing Membrane Proteins from Gas Phase

2019 ◽  
Author(s):  
Matthias Wilm
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Self Assembly ◽  
Protein Function ◽  
Lipid Membranes ◽  
Protein Complexes ◽  
Biological Membranes ◽  
The Self ◽  
In Vitro Systems

1.AbstractMembrane proteins carry out a wide variety of biological functions. The reproduction of specific properties that have evolved over millions of years of biological membranes in a technically controlled environment is of significant interest. Here a method is presented that allows the self-assembly of a macroscopically large, freely transportable membrane with Outer membrane porin G from Escherichia Coli. The technique does not use protein specific characteristics and therefore, could represent a method for the generation of extended layers of membranes with arbitrary membrane protein content. Such in-vitro systems are relevant in the study of membrane-protein function and structure and the self-assembly of membrane-based protein complexes. They might become important for the incorporation of the lipid-membranes in technological devices.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Correlative microscopy in distrubuted (client/server) computing environments: tools for catalyzing the rapid dissemination of information about the cell biology of the human prostate

1995 ◽  
Vol 53 ◽  
pp. 682-683
Author(s):  
A. Hakam ◽  
J.T. Gau ◽  
M.L. Grove ◽  
B.A. Evans ◽  
M. Shuman ◽  
...  
Keyword(s):  
United States ◽  
Cell Biology ◽  
Tissue Bank ◽  
The United States ◽  
Prostate Adenocarcinoma ◽  
Correlative Microscopy ◽  
Client Server ◽  
Critical Elements ◽  
Transplant Organ

Prostate adenocarcinoma is the most common malignant tumor of men in the United States and is the third leading cause of death in men. Despite attempts at early detection, there will be 244,000 new cases and 44,000 deaths from the disease in the United States in 1995. Therapeutic progress against this disease is hindered by an incomplete understanding of prostate epithelial cell biology, the availability of human tissues for in vitro experimentation, slow dissemination of information between prostate cancer research teams and the increasing pressure to “ stretch” research dollars at the same time staff reductions are occurring.To meet these challenges, we have used the correlative microscopy (CM) and client/server (C/S) computing to increase productivity while decreasing costs. Critical elements of our program are as follows:1) Establishing the Western Pennsylvania Genitourinary (GU) Tissue Bank which includes >100 prostates from patients with prostate adenocarcinoma as well as >20 normal prostates from transplant organ donors.

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