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In plant cells, how integral plasma membrane (PM) proteins are degraded in a cargo ubiquitination‐independent manner remains elusive. Here, we studied the degradative pathway of two plant PM proteins: AtLRR84A, a type I integral membrane protein belonging to the leucine‐rich repeat receptor‐like kinase protein family, and OsSCAMP1 (rice secretory carrier membrane protein 1), a tetraspan transmembrane...
Proteomics is a powerful technique for protein identification at large scales. A number of proteomics approaches have been developed to study the steady state composition of intracellular compartments. Here, we report a novel vectorial proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans‐Golgi network (TGN). This strategy is based on the covalent modification of the plasma membrane proteome with a membrane impermeable benzylguanine derivative. Benzylguanine‐tagged plasma membrane proteins that are subsequently targeted to the retrograde route are covalently captured by a TGN‐localized SNAP‐tagged fusion protein, which allows for their identification. The approach was validated step‐by‐step using a well explored retrograde cargo protein, the B‐subunit of Shiga toxin. It was then extended to the proteomics format. Among other hits we found one of the historically first identified cargo proteins that undergo retrograde transport, which further validated our approach. Most of the other hits were kinases, receptors or transporters. In conclusion, we have pioneered a vectorial proteomics approach that complements traditional methods for the study of retrograde protein trafficking. This approach is of generic nature and could in principle be extended to other endocytic pathways.
STIM1 is a core component of the store‐operated Ca2+‐entry channel involved in Ca2+‐signaling with an important role in the activation of immune cells and many other cell types. In response to cell activation, STIM1 protein senses low Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) and activates the channel protein Orai1 in the plasma membrane by direct physical contact. The related...
Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin‐based movement of large protein‐aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic...
Electron tomography (ET) is an indispensable high‐resolution tool for three dimensional (3D) imaging in cell biology. When applied to immuno‐labeled cells, ET can provide essential insights in both the cellular architecture and the dynamics. Current protocols for 3D immuno‐labeling of intracellular antigens include permeabilization steps that cause random, extensive cell membrane disruption. This permeabilization results in a poor cell ultrastructure, limiting the usefulness of the specimens for high‐resolution studies. Here we describe a novel method, based on a well‐controlled permeabilization by targeted laser cell perforation, that allows for the 3D immuno‐localization of cytoplasmic antigens in cultured cells. The approach is unique since it is applicable to both chemically and cryo‐fixed cells and leads to a superior ultrastructural preservation for electron microscopy and tomography.
The conserved oligomeric Golgi (COG) complex co‐ordinates retrograde vesicle transport within the Golgi. These vesicles maintain the distribution of glycosylation enzymes between the Golgi's cisternae, and therefore COG is intimately involved in glycosylation homeostasis. Recent years have greatly enhanced our knowledge of COG's composition, protein interactions, cellular function and most recently also its structure. The emergence of COG‐dependent human glycosylation disorders gives particular relevance to these advances. The structural data have firmly placed COG in the family of multi‐subunit tethering complexes that it shares with the exocyst, Dsl1 and Golgi-associated retrograde protein (GARP) complexes. Here, we review our knowledge of COG's involvement in vesicle tethering at the Golgi. In particular, we consider what this knowledge may add to our molecular understanding of vesicle tethering and how it impacts on the fine tuning of Golgi function, most notably glycosylation....
Peroxisomes and mitochondria show a much closer interrelationship than previously anticipated. They co‐operate in the metabolism of fatty acids and reactive oxygen species, but also share components of their fission machinery. If peroxisomes – like mitochondria – also fuse in mammalian cells is a matter of debate and was not yet systematically investigated. To examine potential peroxisomal fusion...
The DSL1 complex is a conserved tethering complex at the endoplasmic reticulum that recognizes Golgi‐derived COPI vesicles and hands them over to the fusion machinery. The DSL1 complex is the simplest tethering complex of the complexes associated with tethering containing helical rods (CATCHR) family. CATCHR tethering complexes play a role at compartments along the exocytic and endocytic pathways. In this review, different functions of the DSL1 complex are discussed, some open questions with the seemingly straightforward picture are pointed out and alternative functions of the DSL1 complex members are mentioned....
Nuclear transport is mediated by transport factors, including the importin β family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin α/β‐mediated import of classical nuclear localization signal (cNLS) – containing cargo is more efficient than other Ran‐dependent import pathways that do not require importin α. In...
During budding of yeast cells peroxisomes are distributed over mother cell and bud, a process that involves the myosin motor protein Myo2p and the peroxisomal membrane protein Inp2p. Here, we show that Pex19p, a peroxin implicated in targeting and complex formation of peroxisomal membrane proteins, also plays a role in peroxisome partitioning. Binding studies revealed that Pex19p interacts with the...
Two‐pore channels (TPCs) constitute a family of endolysosomal cation channels with functions in Ca2+ signaling. We used a mutational analysis to investigate the role of channel domains for the trafficking of the Arabidopsis TPC1 to the tonoplast, a process that is generally not well understood in plants. The results show that the soluble C‐terminus was not essential for targeting but for channel function, while further C‐terminal truncations of two or more transmembrane domains impaired protein trafficking. An N‐terminal dileucine motif (EDPLI) proved to be critical for vacuolar targeting of TPC1, which was independent of the adaptor protein AP‐3. Deletion or mutation of this sorting motif, which is conserved among TPCs caused redirection of the protein transport to the plasma membrane. An N‐terminal region with a predicted α‐helical structure was shown to support efficient vacuolar trafficking and was essential for TPC1 function. Similar to their localization in mammalian endosomes and lysosomes, MmTPC1 and MmTPC2 were targeted to small organelles and the membrane of the lytic vacuole, respectively, when expressed in plant cells. These results shed new light on the largely uncharacterized sorting signals of plant tonoplast proteins and reveal similarities between the targeting machinery of plants and mammals.
Activity‐dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode during intense neuronal activity. The dephosphorylation of Ser774 on dynamin I is essential for triggering of ADBE, as is its subsequent rephosphorylation by glycogen synthase kinase 3 (GSK3). We show that in primary cultures of cerebellar granule neurons the protein kinase Akt phosphorylates GSK3 during intense neuronal activity, ensuring that GSK3 is inactive during intense stimulation to aid dynamin I dephosphorylation. Furthermore, when a constitutively active form of Akt was overexpressed in primary neuronal cultures, ADBE was inhibited with no effect on clathrin‐mediated endocytosis. Thus Akt has two major regulatory roles (i) to ensure efficient dynamin I dephosphorylation via acute activity‐dependent inhibition of GSK3 and (ii) to negatively regulate ADBE when activated in the longer term. This is the first demonstration of a role for Akt in SV recycling and suggests a key role for this protein kinase in modulating synaptic strength during elevated neuronal activity.
The exocyst complex is an evolutionarily conserved multisubunit protein complex implicated in tethering secretory vesicles to the plasma membrane. Originally identified two decades ago in budding yeast, investigations using several different eukaryotic systems have since made great progress toward determination of the overall structure and organization of the eight exocyst subunits. Studies point to a critical role for the complex as a spatiotemporal regulator through the numerous protein and lipid interactions of its subunits, although a molecular understanding of exocyst function has been challenging to elucidate. Recent progress demonstrates that the exocyst is also important for additional trafficking steps and cellular processes beyond exocytosis, with links to development and disease. In this review, we discuss current knowledge of exocyst architecture, assembly, regulation and its roles in a variety of cellular trafficking pathways....
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