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Oxysterol‐binding protein (OSBP)‐related protein Kes1/ Osh4p is implicated in nonvesicular sterol transfer between membranes in Saccharomyces cerevisiae. However, we found that Osh4p associated with exocytic vesicles that move from the mother cell into the bud, where Osh4p facilitated vesicle docking by the exocyst tethering complex at sites of polarized growth on the plasma membrane. Osh4p formed...
A large number of proteins involved in the biogenesis of yeast endosomes and vacuoles have been identified based on screens that scored for inactivation of proteins. Such screens may, however, miss important regulators of the pathway. Here, we present a visual screen in which we examined the effects on vacuole morphology if any of the 6153 yeast open reading frames was overexpressed. Using a progressive...
Caveolin (CAV) is an essential component of caveolae, cholesterol‐enriched invaginations of the plasma membrane of most mammalian cells. However, CAV is not restricted to plasma membrane caveolae, and pools of CAV are present in myriad intracellular membranes. CAV proteins tightly bind cholesterol and contribute to regulation of cholesterol fluxes and distributions within cells. In this context, we recently showed that CAV1 regulates the poorly understood process controlling mitochondrial cholesterol levels. Cholesterol accumulates in mitochondrial membranes in the absence of CAV1, promoting the organelle's dysfunction with important metabolic consequences for cells and animals. In this article, we suggest a working hypothesis that addresses the role of CAV1 within the homeostatic network that regulates the influx/efflux of mitochondrial cholesterol.
Dynamin (Dyn) is a multidomain and multifunctional GTPase best known for its essential role in clathrin‐mediated endocytosis (CME). Dyn2 mutations have been linked to two human diseases, centronuclear myopathy (CNM) and Charcot‐Marie‐Tooth (CMT) disease. Paradoxically, although Dyn2 is ubiquitously expressed and essential for embryonic development, the disease‐associated Dyn2 mutants are autosomal dominant, but result in slowly progressing and tissue‐specific diseases. Thus, although the cellular defects that cause disease remain unclear, they are expected to be mild. To gain new insight into potential pathogenic mechanisms, we utilized mouse Dyn2 conditional knockout cells combined with retroviral‐mediated reconstitution to mimic both heterozygous and homozygous states and characterized cellular phenotypes using quantitative assays for several membrane trafficking events. Surprisingly, none of the four mutants studied exhibited a defect in CME, but all were impaired in their ability to support p75/neurotrophin receptor export from the Golgi, the raft‐dependent endocytosis of cholera toxin and the clathrin‐independent endocytosis of epidermal growth factor receptor (EGFR). While it will be important to study these mutants in disease‐relevant muscle and neuronal cells, given the importance of neurotrophic factors and lipid rafts in muscle physiology, we speculate that these common cellular defects might contribute to the tissue‐specific diseases caused by a ubiquitously expressed protein.
The Sec24 subunit of the coat protein complex II (COPII) has been implicated in selecting newly synthesized cargo from the endoplasmic reticulum (ER) for delivery to the Golgi. The protozoan parasite, Trypanosoma brucei, contains two paralogs, TbSec24.1 and TbSec24.2, which were depleted using RNA interference in the insect form of the parasite. Depletion of either TbSec24.1 or TbSec24.2 resulted...
MicroRNAs (miRNAs) are negative regulators of gene expression in eukaryotic organisms, whereas small interfering RNAs (siRNAs) guide host‐cell defence against viruses, transposons and transgenes. A key issue in plant biology is whether miRNAs act only in cells in which they are formed, or if, like siRNAs, they also function after passive diffusion or active transportation into other cells. Recent reports show that miRNAs are indeed able to move between plant cells to direct developmental programming of gene expression. In both leaf and root development, miRNAs establish intercellular gradients of gene expression that are essential for cell and tissue differentiation. Gradients in gene expression also play crucial roles in animal development, and there is strong evidence for intercellular movement of miRNAs in animals. Thus, intercellular movement of miRNAs may be crucial to animal developmental biology as well as plants....
Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL)...
By analogy to other axonal proteins, transcytotic delivery following spontaneous endocytosis from the somatodendritic membrane is expected to be essential for polarized distribution of axonal G‐protein coupled receptors (GPCRs). However, possible contribution from constitutive activation, which may also result in constitutive GPCR endocytosis, is poorly known. Using two closely related but differentially...
In order to accurately target the embryo sac and deliver the sperm cells, the pollen tube has to find an efficient path through the pistil and respond to precise directional cues produced by the female tissues. Although many chemical and proteic signals have been identified to guide pollen tube growth, the mechanism by which the tube changes direction in response to these signals is poorly understood...
Non‐integral membrane proteins frequently act as transduction hubs in vital signaling pathways initiated at the plasma membrane (PM). Their biological activity depends on dynamic interactions with the PM, which are governed by their lateral and cytoplasmic diffusion and membrane binding/unbinding kinetics. Accurate quantification of the multiple kinetic parameters characterizing their membrane interaction...
Adaptor protein (AP) complexes are key factors for the spatial and temporal regulation of intracellular trafficking events. Four complexes (AP‐1, ‐2, ‐3, ‐4) are known, among which AP‐4 is only poorly characterized. Recent work suggests a role for AP‐4 in the intracellular trafficking of the β‐amyloid precursor protein and molecular genetics showed that the loss of functional AP‐4 is associated with congenital neuronal disorders of severe cognitive dysfunction. To unravel the molecular mechanisms controlling AP‐4 functions, we established the intracellular expression of recombinant AP‐4 complex. This approach combined with the analysis of mutant complexes allowed us to discover that the epsilon adaptin hinge‐ear region has a function in membrane recruitment of AP‐4. We further show that this process is phosphorylation dependent and involves PP2A‐like protein phosphatases and a staurosporine‐sensitive kinase. Deletion of the residues 839‐871 in the carboxy‐terminal region of the hinge of epsilon adaptin abrogated the membrane/cytosol recycling of AP‐4. As targets of phosphorylation, we identified three serine residues: S847, S868 and S871. We conclude that the terminal hinge region and the appendage of the AP‐4 epsilon subunit are involved in membrane association in a process that is controlled by phosphorylation and dephosphorylation events.
We screened a panel of compounds derived from Exo2—a drug that perturbs post‐Golgi compartments and trafficking in mammalian cells—for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic...
Ubiquitin‐specific protease 33 (USP33) is a deubiquitinase that has been associated with a variety of physiological events. Here, we show the existence of multiple USP33 splice variants and characterize the sub‐cellular localization of endogenous USP33 as well as GFP‐USP33 isoforms 1–3. The localization of USP33 is broadly confined to the secretory pathway, with all variants localizing to endoplasmic reticulum‐associated structures. In addition, GFP‐USP33 variant 3 shows a marked accumulation at the Golgi apparatus. Several deubiquitinases have large insertions within their otherwise highly conserved catalytic domains, the function of which is poorly characterized. Analysis of USP33 reveals a role for two distinct inserts within the catalytic domain. One is required for association with the endoplasmic reticulum, whilst the second is required for membrane association, but can be alternatively spliced (variant 3) to excise eight amino acids, which otherwise suppress Golgi localization. We propose that varying the expression of differentially localized isoforms provides a means to influence the spectrum of substrates encountered by USP33.
pH varies widely among the different intracellular compartments. The establishment and maintenance of a particular pH appears to be critical for proper organellar function. This has been deduced from experiments where intraorganellar pH was altered by means of weak acids or bases, ionophores or inhibitors of the vacuolar H+‐ATPase (V‐ATPase). These manipulations, however, are not specific and simultaneously alter the pH of multiple compartments. As a result, it is difficult to assign their effect to a defined organelle. To circumvent this limitation, we designed and implemented a procedure to selectively manipulate the pH of a compartment of choice, using lysosomes as a model organelle. The approach is based on the targeted and continuous enzymatic generation of weak electrolyte, which enabled us to overcome the high buffering capacity of the lysosomal lumen, without altering the pH of other compartments. We targeted jack‐bean urease to lysosomes and induced the localized generation of ammonia by providing the membrane‐permeant substrate, urea. This resulted in a marked, rapid and fully reversible alkalinization that was restricted to the lysosomal lumen, without measurably affecting the pH of endosomes or of the cytosol. The acute alkalinization induced by urease–urea impaired the activity of pH‐dependent lysosomal enzymes, including cathepsins C and L, without altering endosomal function. This approach, which can be extended to other organelles, enables the analysis of the role of pH in selected compartments, without the confounding effects of global disturbances in pH or vesicular traffic.
Small RNAs with lengths of 20–30 nucleotides, such as microRNAs (miRNAs), play important regulatory roles in various cellular processes. In conventional linear processing pathways, precursors of miRNAs are transported out of the nucleus by the specific nuclear transport receptor, exportin‐5. The exported precursors are further processed and eventually incorporated into the RNA‐induced silencing complex (RISC), which silences the expression of the target genes by posttranscriptional mechanisms in the cytoplasm. Subsequent identification and characterization of P‐element induced wimpy testis (PIWI)‐interacting small RNAs (piRNAs) and endogenous small interfering RNAs (endo‐siRNAs) revealed that the processing mechanisms of these newly emerging small RNAs differ from those of miRNAs. Moreover, cumulative experimental evidence indicates that the nuclear functions of the small RNAs, such as transcriptional gene silencing, could be widespread in divergent species. These observations appended other interesting features in the biogenesis and nucleocytoplasmic transport mechanisms of these small RNAs. In this review, we discuss the mechanisms and biological significance of the intracellular trafficking of small RNAs in animal cells....
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