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Endocytosis is a conserved process across species in which cell surface receptors and lipids are internalized from the plasma membrane. Once internalized, receptors can either be degraded or be recycled back to the plasma membrane. A variety of small GTP‐binding proteins regulate receptor recycling. Despite our familiarity with many of the key regulatory proteins involved in this process, our understanding...
Chediak–Higashi syndrome is an autosomal recessive disorder that affects vesicle morphology. The Chs1/Lyst protein is a member of the BEige And CHediak family of proteins. The absence of Chs1/Lyst gives rise to enlarged lysosomes. Lysosome size is regulated by a balance between vesicle fusion and fission and can be reversibly altered by acidifying the cytoplasm using Acetate Ringer's or by incubating...
The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo‐phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function...
The potassium channel Kv7.1 is expressed in the heart, where it contributes to the repolarization of the cardiac action potential. Additionally, Kv7.1 is expressed in epithelial tissues playing a role in salt and water transport. We recently demonstrated that surface‐expressed Kv7.1 is internalized in response to polarization of the epithelial Madin–Darby canine kidney (MDCK) cell line and that this...
Post‐translational modification by ubiquitination determines intracellular location and fate of numerous proteins, thus impacting a diverse array of physiologic functions. Past dogma has been that ubiquitin was only coupled to substrates by isopeptide bonds to internal lysine residues or less frequently peptide bonds to the N‐terminus. Enigmatically, however, several proteins lacking lysines had been reported to retain ubiquitin‐dependent fates. Resolution of this paradox was afforded by recent observations that ubiquitination of substrates can also occur on cysteine or serine and threonine residues by thio‐ or oxy‐ester bond formation, respectively (collectively called esterification). Although chemically possible, these bonds were considered too labile to be of physiological relevance. In this review we discuss recent evidence for the ubiquitination of protein substrates by esterification and speculate on its mechanism and its physiological importance....
The biogenesis of multivesicular endosomes and the sorting of activated signaling receptors into multivesicular endosomes depend on soluble protein complexes (ESCRT complexes), which transiently interact with the receptor cargo and the endosomal membrane. Previously, it was shown that the transmembrane protein secretory carrier membrane protein (SCAMP) 3, which is present on endosomes, interacts with...
In clathrin‐mediated membrane traffic, clathrin does not bind directly to cargo and instead binds to adaptors that mediate this function. For endocytosis, the main adaptor is the adaptor protein (AP)‐2 complex, but it is uncertain how clathrin contacts AP‐2. Here we tested in human cells the importance of the three binding sites that have been identified so far on the N‐terminal domain (NTD) of clathrin. We find that mutation of each of the three sites on the NTD, alone or in combination, does not block clathrin/AP‐2‐mediated endocytosis in the same way as deletion of the NTD. We report here the fourth and final site on the NTD that is required for clathrin/AP‐2‐mediated endocytic function. Each of the four interaction sites can operate alone to mediate endocytosis. The observed functional redundancy between interaction sites on the NTD explains how productivity of clathrin‐coated vesicle formation is ensured.
It is becoming clear that intracellular signaling events are intimately linked with the membrane transport processes. In addition to the long known role of endocytosis in downregulating plasma membrane receptors, more recent data uncover several sophisticated modes by which endocytosis affects the type and duration of signals. Particularly striking are various roles of endocytic compartments as membrane platforms for compartmentalized assembly or sequestration of specific signaling complexes. Here we review some recent examples illustrating how endosomes may mediate ligand‐stimulated apoptotic signaling and how multivesicular bodies affect Wnt signaling by regulated sequestration of signaling molecules or their secretion in exosomes. We also discuss evidence documenting the involvement of endocytic proteins in the regulation of p53 activity and stability, which suggests a possible cross‐talk between endocytic processes and transcriptional responses....
Quantum dots are bright, photostable fluorophores used extensively to investigate biological processes. In this study, we report that bromocresol green (BCG) at low micromolar concentrations rapidly, efficiently and reversibly quenches the fluorescence of commercial quantum dots having a wide range of functionalities. The broad utility of BCG quenching of quantum dots in cell biology is showed in...
Infection of cells with African swine fever virus (ASFV) can lead to the formation of zipper‐like stacks of structural proteins attached to collapsed endoplasmic reticulum (ER) cisternae. We show that the collapse of ER cisternae observed during ASFV infection is dependent on the viral envelope protein, J13Lp. Expression of J13Lp alone in cells is sufficient to induce collapsed ER cisternae. Collapse was dependent on a cysteine residue in the N‐terminal domain of J13Lp exposed to the ER lumen. Luminal collapse was also dependent on the expression of J13Lp within stacks of ER where antiparallel interactions between the cytoplasmic domains of J13Lp orientated N‐terminal domains across ER cisternae. Cisternal collapse was then driven by disulphide bonds between N‐terminal domains arranged in antiparallel arrays across the ER lumen. This provides a novel mechanism for biogenesis of modified stacks of ER present in cells infected with ASFV, and may also be relevant to cellular processes.
Phosphoinositides play an important role in organelle identity by recruiting effector proteins to the host membrane organelle, thus decorating that organelle with molecular identity. Phosphatidylinositol‐3,5‐bisphos‐ phate [PtdIns(3,5)P2] is a low‐abundance phosphoinositide that predominates in endolysosomes in higher eukaryotes and in the yeast vacuole. Compared to other phosphoinositides such as...
The organization of eukaryotic cells into membrane‐bound compartments must be faithfully sustained for survival of the cell. A subtle equilibrium exists between the degradation and the proliferation of organelles. Commonly, proliferation is initiated by a membrane remodeling process. Here, we dissect the function of proteins driving organelle proliferation in the particular case of peroxisomes. These...
During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal‐targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1‐containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was...
Endosomal sorting is essential for cell homeostasis. Proteins targeted for degradation are retained in the maturing endosome vacuole while others are recycled to the cell surface or sorted to the biosynthetic pathway via tubular transport carriers. Sorting nexin (SNX) proteins containing a BAR (for Bin–Amphiphysin–Rvs) domain are key regulators of phosphoinositide‐mediated, tubular‐based endosomal...
The supramolecular assembly of aquaporin‐4 (AQP4) in orthogonal arrays of particles (OAPs) involves N‐terminus interactions of the M23‐AQP4 isoform. We found AQP4 OAPs in cell plasma membranes but not in endoplasmic reticulum (ER) or Golgi, as shown by: (i) native gel electrophoresis of brain and AQP4‐transfected cells, (ii) photobleaching recovery of green fluorescent protein‐AQP4 chimeras in live cells and (iii) freeze‐fracture electron microscopy (FFEM). We found that AQP4 OAP formation in plasma membranes, but not in the Golgi, was not related to AQP4 density, pH, membrane lipid composition, C‐terminal PDZ domain interactions or α‐syntrophin expression. Remarkably, however, fusion of AQP4‐containing Golgi vesicles with (AQP4‐free) plasma membrane vesicles produced OAPs, suggesting the involvement of plasma membrane factor(s) in AQP4 OAP formation. In investigating additional possible determinants of OAP assembly we discovered membrane curvature‐dependent OAP assembly, in which OAPs were disrupted by extrusion of plasma membrane vesicles to ∼110 nm diameter, but not to ∼220 nm diameter. We conclude that AQP4 supramolecular assembly in OAPs is a post‐Golgi phenomenon involving plasma membrane‐specific factor(s). Post‐Golgi and membrane curvature‐dependent OAP assembly may be important for vesicle transport of AQP4 in the secretory pathway and AQP4‐facilitated astrocyte migration, and suggests a novel therapeutic approach for neuromyelitis optica.
The brain‐spliced isoform of Myosin Va (BR‐MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide‐producing cells. The molecular composition of the protein complex that recruits BR‐MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG‐associated proteins granuphilin‐a/b (Gran‐a/b), BR‐MyoVa and Rab27a, a member of the Rab family of GTPases. Gran‐a/b–BR‐MyoVa interaction is direct, involves regions downstream of the Rab27‐binding domain, and the C‐terminal part of Gran‐a determines exon specificity. MyoVa and Gran‐a/b are partially colocalised on SGs and disruption of Gran‐a/b–BR‐MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient‐stimulated hormone secretion in pancreatic beta‐cells. These results indicate the existence of at least another binding partner of BR‐MyoVa that was identified as rabphilin‐3A (Rph‐3A). BR‐MyoVa–Rph‐3A interaction is also direct and enhanced when secretion is activated. The BR‐MyoVa–Rph‐3A and BR‐MyoVa–Gran‐a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR‐MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.
The assembly of clathrin‐coated vesicles is important for numerous cellular processes, including nutrient uptake and membrane organization. Important contributors to clathrin assembly are four tetrameric assembly proteins, also called adaptor proteins (APs), each of which contains a β subunit. We identified a single β subunit, named β1/2, that contributes to both the AP1 and AP2 complexes of Dictyostelium...
Toxoplasma gondii utilizes specialized secretory organelles called rhoptries to invade and hijack its host cell. Many rhoptry proteins are proteolytically processed at a highly conserved SΦXE site to remove organellar targeting sequences that may also affect protein activity. We have studied the trafficking and biogenesis of a secreted rhoptry metalloprotease with homology to insulysin that we named...
The reorganization of nuclear structures is an important early feature of apoptosis and involves the activity of specific proteases and nucleases. Well‐known is the condensation and fragmentation of chromatin; however, much less is understood about the mechanisms involved in the reorganization of structures from the interchromatin space, such as interchromatin granule clusters (IGCs). In this study,...
Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin‐related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset...
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