Distinct Membrane Domains on Endosomes in the Recycling Pathway Visualized by Multicolor Imaging of Rab4, Rab5, and Rab11

Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.

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Faculty Opinions recommendation of Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1005035.59104 ◽

2002 ◽

Author(s):

Bettina Winckler

Keyword(s):

Membrane Domains ◽

Multicolor Imaging ◽

Recycling Pathway ◽

Distinct Membrane

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Faculty Opinions recommendation of Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1025186.298879 ◽

2005 ◽

Author(s):

Judith S Eisen

Keyword(s):

Growth Cone ◽

Membrane Domains ◽

Distinct Membrane

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Structures ofDrosophila melanogasterRab2 and Rab3 bound to GMPPNP

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x1402617x ◽

2015 ◽

Vol 71 (1) ◽

pp. 34-40 ◽

Cited By ~ 4

Author(s):

Jennifer A. Lardong ◽

Jan H. Driller ◽

Harald Depner ◽

Christoph Weise ◽

Astrid Petzoldt ◽

...

Keyword(s):

Conformational Changes ◽

Intracellular Trafficking ◽

Large Family ◽

Cysteine Residue ◽

Effector Proteins ◽

Rab Proteins ◽

Rab Gtpases ◽

Ras Proteins ◽

Transport Vesicles ◽

Switch Regions

Rab GTPases belong to the large family of Ras proteins. They act as key regulators of membrane organization and intracellular trafficking. Functionally, they act as switches. In the active GTP-bound form they can bind to effector proteins to facilitate the delivery of transport vesicles. Upon stimulation, the GTP is hydrolyzed and the Rab proteins undergo conformational changes in their switch regions. This study focuses on Rab2 and Rab3 fromDrosophila melanogaster. Whereas Rab2 is involved in vesicle transport between the Golgi and the endoplasmatic reticulum, Rab3 is a key player in exocytosis, and in the synapse it is involved in the assembly of the presynaptic active zone. Here, high-resolution crystal structures of Rab2 and Rab3 in complex with GMPPNP and Mg2+are presented. In the structure of Rab3 a modified cysteine residue is observed with an enigmatic electron density attached to its thiol function.

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Calnexin revealed as an ether-a-go-go chaperone by getting mutant worms up and going

The Journal of General Physiology ◽

10.1085/jgp.201812068 ◽

2018 ◽

Vol 150 (8) ◽

pp. 1059-1061

Author(s):

Jonathan T. Pierce

Keyword(s):

Ion Channels ◽

Dynamic Control ◽

Cell Types ◽

K Channel ◽

Membrane Domains ◽

Excitable Cells ◽

Patch Clamp Recording ◽

Cell Excitability ◽

Distinct Membrane

The role of ion channels in cell excitability was first revealed in a series of voltage clamp experiments by Hodgkin and Huxley in the 1950s. However, it was not until the 1970s that patch-clamp recording ushered in a revolution that allowed physiologists to witness how ion channels flicker open and closed at angstrom scale and with microsecond resolution. The unexpectedly tight seal made by the patch pipette in the whole-cell configuration later allowed molecular biologists to suck up the insides of identified cells to unveil their unique molecular contents. By refining these techniques, researchers have scrutinized the surface and contents of excitable cells in detail over the past few decades. However, these powerful approaches do not discern which molecules are responsible for the dynamic control of the genesis, abundance, and subcellular localization of ion channels. In this dark territory, teams of unknown and poorly understood molecules guide specific ion channels through translation, folding, and modification, and then they shuttle them toward and away from distinct membrane domains via different subcellular routes. A central challenge in understanding these processes is the likelihood that these diverse regulatory molecules may be specific to ion channel subtypes, cell types, and circumstance. In work described in this issue, Bai et al. (2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812025) begin to shed light on the biogenesis of UNC-103, a K+ channel found in Caenorhabditis elegans.

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Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

mBio ◽

10.1128/mbio.01800-17 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 13

Author(s):

Michael G. Connor ◽

Amanda R. Pulsifer ◽

Donghoon Chung ◽

Eric C. Rouchka ◽

Brian K. Ceresa ◽

...

Keyword(s):

Yersinia Pestis ◽

Host Factors ◽

Endocytic Recycling ◽

Recycling Endosome ◽

Independent Manner ◽

Genome Wide ◽

Type Three Secretion System ◽

A Genome ◽

Recycling Pathway ◽

Bacterial Replication

ABSTRACTYersinia pestishas evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis,Y. pestisprevents phagolysosome maturation and establishes a modified compartment termed theYersinia-containing vacuole (YCV).Y. pestisactively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requiresY. pestisinteractions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required forY. pestissurvival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival ofY. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated thatY. pestisactively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV byY. pestisto resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered thatY. pestisdisrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence thatY. pestistargets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV.IMPORTANCEYersinia pestiscan infect and survive within macrophages. However, the mechanisms that the bacterium use to subvert killing by these phagocytes have not been defined. To provide a better understanding of these mechanisms, we used an RNAi approach to identify host factors required for intracellularY. pestissurvival. This approach revealed that the host endocytic recycling pathway is essential forY. pestisto avoid clearance by the macrophage. We further demonstrate thatY. pestisremodels the phagosome to resemble a recycling endosome, allowing the bacterium to avoid the normal phagolysosomal maturation pathway. Moreover, we show that infection withY. pestisdisrupts normal recycling in the macrophage and that disruption is required for bacterial replication. These findings provide the first evidence thatY. pestistargets the host endocytic recycling pathway in order to evade killing by macrophages.

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Roco Proteins and the Parkinson’s Disease-Associated LRRK2

International Journal of Molecular Sciences ◽

10.3390/ijms19124074 ◽

2018 ◽

Vol 19 (12) ◽

pp. 4074 ◽

Cited By ~ 5

Author(s):

Jingling Liao ◽

Quyen Hoang

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

G Proteins ◽

Conformational Changes ◽

Effector Proteins ◽

Cellular Processes ◽

Current State ◽

Specific Effector ◽

Unique Domain ◽

Small G Proteins

Small G-proteins are structurally-conserved modules that function as molecular on-off switches. They function in many different cellular processes with differential specificity determined by the unique effector-binding surfaces, which undergo conformational changes during the switching action. These switches are typically standalone monomeric modules that form transient heterodimers with specific effector proteins in the ‘on’ state, and cycle to back to the monomeric conformation in the ‘off’ state. A new class of small G-proteins called “Roco” was discovered about a decade ago; this class is distinct from the typical G-proteins in several intriguing ways. Their switch module resides within a polypeptide chain of a large multi-domain protein, always adjacent to a unique domain called COR, and its effector kinase often resides within the same polypeptide. As such, the mechanisms of action of the Roco G-proteins are likely to differ from those of the typical G-proteins. Understanding these mechanisms is important because aberrant activity in the human Roco protein LRRK2 is associated with the pathogenesis of Parkinson’s disease. This review provides an update on the current state of our understanding of the Roco G-proteins and the prospects of targeting them for therapeutic purposes.

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A model for Rab GTPase localization

Biochemical Society Transactions ◽

10.1042/bst0330627 ◽

2005 ◽

Vol 33 (4) ◽

pp. 627-630 ◽

Cited By ~ 48

Author(s):

S. Pfeffer

Keyword(s):

Steady State ◽

Cellular Localization ◽

Rab Gtpase ◽

Rab Proteins ◽

Rab Gtpases ◽

Macromolecular Assemblies ◽

Complex Interactions ◽

Membrane Compartment ◽

Membrane Bound ◽

Distinct Membrane

The human genome encodes almost 70 Rab GTPases. These proteins are C-terminally geranylgeranylated and are localized to the surfaces of distinct membrane-bound compartments in eukaryotic cells. This mini review presents a working model for how Rabs achieve and maintain their steady-state localizations. Data from a number of laboratories suggest that Rabs participate in the generation of macromolecular assemblies that generate functional microdomains within a given membrane compartment. Our data suggest that these complex interactions are important for the cellular localization of Rab proteins at steady state.

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Rabenosyn-5, a Novel Rab5 Effector, Is Complexed with Hvps45 and Recruited to Endosomes through a Fyve Finger Domain

The Journal of Cell Biology ◽

10.1083/jcb.151.3.601 ◽

2000 ◽

Vol 151 (3) ◽

pp. 601-612 ◽

Cited By ~ 222

Author(s):

Erik Nielsen ◽

Savvas Christoforidis ◽

Sandrine Uttenweiler-Joseph ◽

Marta Miaczynska ◽

Frederique Dewitte ◽

...

Keyword(s):

Early Endosome ◽

Effector Proteins ◽

Endosomal Trafficking ◽

Membrane Domains ◽

Phosphatidylinositol 3 Kinase ◽

Early Endosomes ◽

Site Of Action ◽

Endosomal Membranes ◽

Novel Protein ◽

Phosphatidylinositol 3

Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase–dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

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The structure of the periplast components and their association with the plasma membrane in a cryptomonad flagellate

Canadian Journal of Botany ◽

10.1139/b87-141 ◽

1987 ◽

Vol 65 (5) ◽

pp. 1019-1026 ◽

Cited By ~ 10

Author(s):

Richard Wetherbee ◽

David R. A. Hill ◽

Steven J. Brett

Keyword(s):

Surface Layer ◽

Plasma Membrane ◽

Thin Sheet ◽

Subsequent Development ◽

Membrane Domains ◽

Cytoplasmic Surface ◽

Plate Size ◽

Degree Of Association ◽

Distinct Membrane ◽

Crystalline Array

The periplast of Cryptomonas sp. θ covers most of the cell surface and is composed of the plasma membrane sandwiched between inner and surface periplast components. The surface periplast component is tightly appressed to the plasma membrane and consists of irregularly shaped plates composed of subunits organized into a crystalline array. Noncrystalline material distinguishes plate borders, and changes in plate size and (or) shape may result from the addition or subtraction of subunits at the borders. The inner periplast component is difficult to discern, but normally appears as a thin sheet of material appressed to the cytoplasmic surface of the plasma membrane. The inner periplast component is not closely associated with the plasma membrane at the positions corresponding to plate borders in the overlying surface periplast component. Both periplast components end at the entrance to the vestibulum, which together with the gullet is covered by a surface layer of heptagonal "rosette scales." The size and shape of the surface periplast plates, as well as the degree of association of the inner periplast sheet with the plasma membrane, are mirrored in the P and, to a lesser degree, E fracture faces of the plasma membrane. The presence of distinct membrane domains suggests the plasma membrane may be directly involved in the assembly and subsequent development of the periplast layers.

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