Regulation Of Protein Sorting Research Articles

A new vesicle trafficking regulator CTL1 plays a crucial role in ion homeostasis.

Ion homeostasis is essential for plant growth and environmental adaptation, and maintaining ion homeostasis requires the precise regulation of various ion transporters, as well as correct root patterning. However, the mechanisms underlying these processes remain largely elusive. Here, we reported that a choline transporter gene, CTL1, controls ionome homeostasis by regulating the secretory trafficking of proteins required for plasmodesmata (PD) development, as well as the transport of some ion transporters. Map-based cloning studies revealed that CTL1 mutations alter the ion profile of Arabidopsis thaliana. We found that the phenotypes associated with these mutations are caused by a combination of PD defects and ion transporter misregulation. We also established that CTL1 is involved in regulating vesicle trafficking and is thus required for the trafficking of proteins essential for ion transport and PD development. Characterizing choline transporter-like 1 (CTL1) as a new regulator of protein sorting may enable researchers to understand not only ion homeostasis in plants but also vesicle trafficking in general.

TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion.

SummaryCollagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.

Quantitative Phosphoproteomics of Cytotoxic T Cells to Reveal Protein Kinase D 2 Regulated Networks

The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages.

Vertebrate galectins: endogenous regulators of ionotropic glutamate receptors?

Lectins are proteins that bind specific carbohydrate residues on glycoproteins and glycolipids. In many cases, such binding alters the functional properties of the targeted protein or lipid and triggers a change in cell physiology. First characterized in plants, lectins with diverse carbohydrate specificity have subsequently been identified in animals, where they serve a variety of different functions including both intracellular regulation of protein sorting and extracellular interactions with carbohydrate moieties on the cell surface and in the extracellular matrix (Dodd & Drickamer, 2001). Both plant and animal lectins typically exist as dimers or multimers with their biological action being dependent on, or enhanced by, the ability to form cross links between protein subunits. In the case of ionotropic glutamate receptors (iGluRs) early work with plant lectins demonstrated a potentiating action on agonist-evoked currents recorded in insect and vertebrate cells. Vertebrates express three distinct iGluR families, but only α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) subtypes exhibit strong direct modulation by lectins while N-methyl-d-aspartate (NMDA) receptors are relatively unaffected. Brief exposure to wheat germ agglutinin, concanavalin A (ConA) or isolectin B4, with specificity for N-acetylglucosamine, mannose and β-galactosides, respectively, increases the amplitude of steady-state currents evoked via KA and/or AMPA receptors (Mayer & Vyklicky, 1989; Huettner, 1990). Now, a study from Geoff Swanson's lab in this issue of The Journal of Physiology (Copits et al. 2014) investigates the ability of vertebrate galectins, including recombinant human galectin-1, and congerin I and II from the conger eel, to regulate native and recombinant AMPA and KA receptors. Galectins bind specifically to β-galactoside glycoconjugates and the three galectins studied by Copits et al. (2014) can each form a bivalent homodimer. In contrast to tetravalent ConA, which substantially increases currents mediated by most AMPA and KA receptor isoforms, galectin application produced a remarkable diversity of effects on recombinant iGluRs depending on the specific subunits that were expressed and on which galectin was applied. For example, congerin caused a substantial slowing of AMPA receptor desensitization with an increase in steady-state current in cells expressing homomeric GluA4 and some of the cells expressing GluA1 whereas human galectin-1 had a greater effect on GluA1 than GluA4. In addition, congerin, but not galectin-1, reduced peak current with little change in kinetics in cells that expressed homomeric GluA2 or the GluA1/GluA2 heteromeric combination. Similarly, both eel and human galectins elicited a significantly greater slowing of desensitization in homomeric GluK2 KA receptors than homomeric GluK1 or heteromeric GluK2/GluK5 combinations. Modulation persisted in heterologous cells that were co-transfected with AMPA or KA receptor subunits together with their auxiliary subunits stargazin or Neto2, respectively. Mechanistically, galectin modulation of GluK2 was reduced by drugs that interfere with oligosaccharide processing and prevented by point mutations that eliminate three essential glycosylation sites at the interface between the receptor's amino terminal and ligand binding domains (Everts et al. 1999). In addition, GluK2 modulation was enhanced by linkers that stabilized the recombinant galectin as an obligate dimer, which is consistent with the idea that iGluR modulation involves crosslinking subunits via their attached glycoconjugates. In future work, it would be interesting to determine whether galectin–3, which can assemble into a pentameric oligomer, might elicit even stronger modulation. When tested on neuronal AMPA receptors in cultures prepared from rat hippocampus, congerin failed to change the amplitude or time course of agonist-evoked or spontaneous excitatory synaptic currents, possibly owing to additional modifications of glycoconjugates in central neurons that render them resistant to galectins or, alternatively, prior occupation of the binding site by endogenous lectins produced in the cultures that occlude the binding of acutely applied exogenous galectin. On the other hand, both congerin and human galectin-1 slowed desensitization of KA receptor-mediated currents recorded in freshly dissociated mouse sensory neurons, cells that are known to express galectin-1 and -3 as well as β-galactoside glycoconjugates (Jessell et al. 1990). Thus, Swanson and colleagues (Copits et al. 2014) have established the potential for native receptors to be modulated by endogenous lectins, but much work remains to determine where and when such regulation actually occurs. Mice lacking galectin-1 are viable but exhibit changes in primary afferent morphology and sensitivity (McGraw et al. 2005) as well as resistance to death of central neurons subsequent to pilocarpine-induced seizures (Bischoff et al. 2012). Whether or not these phenotypes involve alteration in iGluR modulation remains to be determined, but the availability of both galectin–1 and galectin–3 knock-outs should aid in unravelling their potential role in regulating excitatory amino acid receptors.

Erratum: COPII and the regulation of protein sorting in mammals

Nat. Cell Biol. 14, 20–28 (2012); published online 22 December 2011; corrected after print 6 January 2012 In the version of this review initially published online and in print, the key in figure 2 was incorrect. The grey dots should represent Sec23–24, the blue lines should represent Sec13–31 and the green dots should represent TRAPPI.

COPII and the regulation of protein sorting in mammals

Secretory proteins are transported to the Golgi complex in vesicles that bud from the endoplasmic reticulum. The cytoplasmic coat protein complex II (COPII) is responsible for cargo sorting and vesicle morphogenesis. COPII was first described in Saccharomyces cerevisiae, but its basic function is conserved throughout all eukaryotes. Nevertheless, the COPII coat has adapted to the higher complexity of mammalian physiology, achieving more sophisticated levels of secretory regulation. In this review we cover aspects of mammalian COPII-mediated regulation of secretion, in particular related to the function of COPII paralogues, the spatial organization of cargo export and the role of accessory proteins.

UMA and MABP domains throw light on receptor endocytosis and selection of endosomal cargoes

Interactions of the ESCRT complexes are critical for endosomal trafficking. We identify two domains with potential significance for this process. The MABP domain present in metazoan ESCRT-I/MVB12 subunits, Crag, a regulator of protein sorting, and bacterial pore-forming proteins might mediate novel membrane interactions in trafficking. The UBAP1-MVB12-associated UMA domain found in MVB12 and UBAP1 defines a novel adaptor that might recruit diverse targets to ESCRT-I.Contact: aravind@ncbi.nlm.nih.govSupplementary information: Supplementary data are available at ftp://ftp.ncbi.nih.gov/pub/aravind/UMA/MVB12.html.

Sorting through the Cell Biology of Alzheimer's Disease: Intracellular Pathways to Pathogenesis

During the first 100 years of Alzheimer's disease research, this devastating and intractable disorder has been characterized at the clinical, histological, and molecular levels. Nevertheless, many key mechanistic questions remain unanswered. Here we will emphasize the importance of the cell biology of Alzheimer's disease, reviewing the relevant literature that has expanded our mechanistic understanding, with a particular focus on pathways regulating protein sorting. Accumulated evidence indicates that sorting pathways may be uniquely vulnerable to disease pathogenesis, and recent studies have begun to reveal disease-related defects in the regulation of protein sorting.

Regulation of protein sorting at the TGN by plasma membrane receptor activation.

We show that in the rat basophilic leukemia cell line RBL, the physiological stimulation of the IgE receptor or direct activation of PKC leads to the missorting of proteins to the plasma membrane, diverting them from their normal intracellular destination. This is demonstrated for two classes of proteins that are normally targeted to the secretory lysosomes via completely different mechanisms, i.e. proteoglycans and the aspartic protease cathepsin D. In the latter case, normal processing of the enzyme is also affected, leading to secretion of the immature form of cathepsin. The present study shows how completely different sorting mechanisms, such as those for delivering proteoglycans and cathepsin D to secretory lysosomes, might share common regulatory signals and are similarly affected when the levels of these signals are perturbed. Finally, protein kinase C appears to be a major player in the signal transduction pathways, leading to proteoglycan and cathepsin D missorting.

Lipids, lipid domains and lipid–protein interactions in endocytic membrane traffic

It has long been known that lipids are responsible for the structural integrity of biological membranes, and confer specific dynamic properties to the bilayer. However, they have often been considered as passive components of the membrane, beyond their well–known function as second messengers in signal transduction pathways. Evidence has been accumulating that lipids are sorted along the routes of membrane traffic, and that they are directly involved in the regulation of protein sorting and membrane flow. In this review, we will discuss recent studies on the organization and possible functions of lipids in the endocytic pathway.

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.