scholarly journals A shared pathway of exosome biogenesis operates at plasma and endosome membranes

2019 ◽  
Author(s):  
Francis K. Fordjour ◽  
George G. Daaboul ◽  
Stephen J. Gould
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Mechanistic Explanation ◽  
Simple Explanation ◽  
Common Pathway ◽  
Exosome Biogenesis ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
Targeting Signal ◽  
Membrane Bound

AbstractEukaryotic cells secrete exosomes, which are small (~30-200 nm dia.), single membrane-bound organelles that transmit signals and molecules to other cells. Exosome-mediated signaling contributes to diverse physiological and disease processes, rendering their biogenesis of high biomedical importance. The prevailing hypothesis is that exosomes bud exclusively at endosome membranes and are released only upon endosome fusion with the plasma membrane. Here we tested this hypothesis by examining the intracellular sorting and exosomal secretion of the exosome cargo proteins CD63, CD9, and CD81. We report here that CD9 and CD81 are both localized to the plasma membrane and bud >5-fold more efficiently than endosome-localized CD63. Furthermore, we show that redirecting CD63 from endosomes to the plasma membrane by mutating its endocytosis signal (CD63/Y235A) increased its exosomal secretion ~6-fold, whereas redirecting CD9 to endosomes by adding an endosome targeting signal (CD9/YEVM) reduced its exosomal secretion ~5-fold. These data demonstrate that the plasma membrane is a major site of exosome biogenesis, and more importantly, that cells possess a common pathway for exosome protein budding that operates at both plasma and endosome membranes. Using a combination of single-particle interferometry reflectance (SPIR) imaging and immunofluorescence (IF) microscopy, we also show that variations in exosome composition are controlled by differential intracellular protein trafficking rather than by separate mechanisms of exosome biogenesis. This new view of exosome biogenesis offers a simple explanation for the pronounced compositional heterogeneity of exosomes and a validated roadmap for exosome engineering.SummaryThis study of exosome cargo protein budding reveals that cells use a common pathway for budding exosomes from plasma and endosome membranes, providing a new mechanistic explanation for exosome heterogeneity and a rational roadmap for exosome engineering.

scholarly journals The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo

2004 ◽  
Vol 165 (1) ◽  
pp. 135-144 ◽  
Author(s):  
Rebecca Dunn ◽  
Deborah A. Klos ◽  
Adam S. Adler ◽  
Linda Hicke
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Trafficking ◽  
C2 Domain ◽  
Intact Cells ◽  
Membrane Interaction ◽  
Cargo Protein ◽  
Membrane Protein Trafficking ◽  
Cargo Proteins

Ubiquitin ligases of the Nedd4 family regulate membrane protein trafficking by modifying both cargo proteins and the transport machinery with ubiquitin. Here, we investigate the role of the yeast Nedd4 homologue, Rsp5, in protein sorting into vesicles that bud into the multivesicular endosome (MVE) en route to the vacuole. A mutant lacking the Rsp5 C2 domain is unable to ubiquitinate or sort biosynthetic cargo into MVE vesicles, whereas endocytic cargo is ubiquitinated and sorted efficiently. The C2 domain binds specifically to phosphoinositides in vitro and is sufficient for localization to membranes in intact cells. Mutation of a lysine-rich patch on the surface of the C2 domain abolishes membrane interaction and disrupts sorting of biosynthetic cargo. Translational fusion of ubiquitin to a biosynthetic cargo protein alleviates the requirement for the C2 domain in its MVE sorting. These results demonstrate that the C2 domain specifies Rsp5-dependent ubiquitination of endosomal cargo and suggest that Rsp5 function is regulated by membrane phosphoinositides.

scholarly journals Microtubule-dependent endosomal sorting of clathrin-independent cargo by Hook1

2013 ◽  
Vol 201 (2) ◽  
pp. 233-247 ◽  
Author(s):  
Lymarie Maldonado-Báez ◽  
Nelson B. Cole ◽  
Helmut Krämer ◽  
Julie G. Donaldson
Keyword(s):  
Plasma Membrane ◽  
Major Histocompatibility Complex ◽  
Cell Spreading ◽  
Complex Class ◽  
Present Evidence ◽  
Endosomal Sorting ◽  
Histocompatibility Complex ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
Bulk Membrane

Many plasma membrane (PM) proteins enter cells nonselectively through clathrin-independent endocytosis (CIE). Here, we present evidence that cytoplasmic sequences in three CIE cargo proteins—CD44, CD98, and CD147—were responsible for the rapid sorting of these proteins into endosomal tubules away from endosomes associated with early endosomal antigen 1 (EEA1). We found that Hook1, a microtubule- and cargo-tethering protein, recognized the cytoplasmic tail of CD147 to help sort it and CD98 into Rab22a-dependent tubules associated with recycling. Depletion of Hook1 from cells altered trafficking of CD44, CD98, and CD147 toward EEA1 compartments and impaired the recycling of CD98 back to the PM. In contrast, another CIE cargo protein, major histocompatibility complex class I, which normally traffics to EEA1 compartments, was not affected by depletion of Hook1. Loss of Hook1 also led to an inhibition of cell spreading, implicating a role for Hook1 sorting of specific CIE cargo proteins away from bulk membrane and back to the PM.

scholarly journals Uncovering targeting priority to yeast peroxisomes using an in-cell competition assay

2020 ◽  
Vol 117 (35) ◽  
pp. 21432-21440
Author(s):  
Mira Rosenthal ◽  
Eyal Metzl-Raz ◽  
Jérôme Bürgi ◽  
Eden Yifrach ◽  
Layla Drwesh ◽  
...  
Keyword(s):  
Cell Types ◽  
Competition Assay ◽  
Cell Competition ◽  
Cargo Protein ◽  
Eukaryotic Proteins ◽  
Cargo Proteins ◽  
Metabolic Conditions ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Binding Interfaces

Approximately half of eukaryotic proteins reside in organelles. To reach their correct destination, such proteins harbor targeting signals recognized by dedicated targeting pathways. It has been shown that differences in targeting signals alter the efficiency in which proteins are recognized and targeted. Since multiple proteins compete for any single pathway, such differences can affect the priority for which a protein is catered. However, to date the entire repertoire of proteins with targeting priority, and the mechanisms underlying it, have not been explored for any pathway. Here we developed a systematic tool to study targeting priority and used the Pex5-mediated targeting to yeast peroxisomes as a model. We titrated Pex5 out by expressing high levels of a Pex5-cargo protein and examined how the localization of each peroxisomal protein is affected. We found that while most known Pex5 cargo proteins were outcompeted, several cargo proteins were not affected, implying that they have high targeting priority. This priority group was dependent on metabolic conditions. We dissected the mechanism of priority for these proteins and suggest that targeting priority is governed by different parameters, including binding affinity of the targeting signal to the cargo factor, the number of binding interfaces to the cargo factor, and more. This approach can be modified to study targeting priority in various organelles, cell types, and organisms.

Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

1977 ◽  
Vol 38 (03) ◽  
pp. 0630-0639 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Early Stage ◽  
Adenyl Cyclase ◽  
Cellular Component ◽  
Primary Event ◽  
Rabbit Platelets ◽  
Membrane Bound

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

scholarly journals Adaptor Protein CD2AP and L-type Lectin LMAN2 Regulate Exosome Cargo Protein Trafficking through the Golgi Complex

2016 ◽  
Vol 291 (49) ◽  
pp. 25462-25475 ◽  
Author(s):  
Sang-Ho Kwon ◽  
Sekyung Oh ◽  
Marisa Nacke ◽  
Keith E. Mostov ◽  
Joshua H. Lipschutz
Keyword(s):  
Golgi Complex ◽  
Protein Trafficking ◽  
Adaptor Protein ◽  
Cargo Protein

scholarly journals PROBLEMS ASSOCIATED WITH THE DEMONSTRATION BY LEAD METHODS OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN RESIDENT PERITONEAL MACROPHAGES AND EXUDATE MONOCYTES OF THE GUINEA PIG

1973 ◽  
Vol 21 (5) ◽  
pp. 488-498 ◽  
Author(s):  
R. E. POELMANN ◽  
W. T. DAEMS ◽  
E. J. VAN LOHUIZEN
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Microscopic Study ◽  
Heterogeneous Nucleation ◽  
Adenosine Triphosphatase ◽  
Peritoneal Macrophages ◽  
Electron Microscopic ◽  
Adenosine Triphosphatase Activity ◽  
Eosinophilic Granulocytes ◽  
Membrane Bound

This cytochemical and electron microscopic study on peritoneal macrophages of the guinea pig has raised doubts concerning the validity of lead methods for the demonstration of plasma membrane-bound adenosine triphosphatase activity. The problems encountered are inherent in the use of lead ions as a capture reagent. The nonenzymatically formed precipitates reflect sites of heterogeneous nucleation specific for certain kinds of cells, e.g., resident peritoneal macrophages, eosinophilic granulocytes and, to a lesser degree, exudate monocytes. This type of precipitation is also catalyzed on the surface of nonbiologic matrices such as latex particles. Enzymatic processes may well occur, but they cannot be distinguished from nonenzymatic processes.

scholarly journals A constitutive, plasma-membrane bound β-glucosidase inTrichoderma reesei

1986 ◽  
Vol 34 (3) ◽  
pp. 291-295 ◽  
Author(s):  
Claudio Umile ◽  
Christian P. Kubicek
Keyword(s):  
Plasma Membrane ◽  
Membrane Bound
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