Endocytosis in plants: Peculiarities and roles in the regulated trafficking of plant metal transporters

The removal of transmembrane proteins from the plasma membrane via endocytosis has emerged as powerful strategy in the regulation of receptor signalling and molecule transport. In the last decade, IRON‐REGULATED TRANSPORTER1 (IRT1) has been established as one of the key plant model proteins for studying endomembrane trafficking. The use of IRT1 and additional other metal transporters has uncovered novel factors involved in plant endocytosis and facilitated a better understanding of the role of endocytosis in the fine balancing of plant metal homoeostasis. In this review, we outline the specifics of plant endocytosis compared to what is known in yeast and mammals, and based on several examples, we demonstrate how studying metal transport has contributed to extending our knowledge of endocytic trafficking by shedding light on novel regulatory mechanisms and factors.     

Viruses customize autophagy protein for efficient viral entry

While the cell imposes multiple barriers to virus entry, enveloped viruses are remarkably still able to gain entry to their cellular hosts by hitchhiking and remodeling the endomembrane system to traffic within, and eventually escape from, endosomal organelles for their genome release. Elucidating viral entry mechanisms and their interaction with the host trafficking network is necessary for antiviral therapy. Here, we focus on the use of host autophagy molecular factors during the entry of prototypic negative-stranded RNA viruses, and highlight recent progress in our understanding of the role of one such factor, UVRAG, in both viral and cellular endocytic membrane trafficking and fusion events.     

Longin and GAF Domains: Structural Evolution and Adaptation to the Subcellular Trafficking Machinery

Endomembrane trafficking is one of the most prominent cytological features of eukaryotes. Given their widespread distribution and specialization, coiled‐coil domains, coatomer domains, small GTPases and Longin domains are considered primordial ‘building blocks’ of the membrane trafficking machineries. Longin domains are conserved across eukaryotes and were likely to be present in the Last Eukaryotic Common Ancestor. The Longin fold is based on the α‐β‐α sandwich architecture and a unique topology, possibly accounting for the special adaptation to the eukaryotic trafficking machinery. The ancient P er A RNT S im (PAS) and cG MP‐specific phosphodiesterases, A denylyl cyclases and F hlA (GAF) family domains show a similar architecture, and the identification of prokaryotic counterparts of GAF domains involved in trafficking provides an additional connection for the endomembrane system back into the pre‐eukaryotic world. Proteome‐wide, comparative bioinformatic analyses of the domains reveal three binding regions (A, B and C) mediating either specific or conserved protein–protein interactions. While the A region mediates intra‐ and inter‐molecular interactions, the B region is involved in binding small GTPases, thus providing an evolutionary connection among major building blocks in the endomembrane system. Finally, we propose that the peculiar interaction surface of the C region of the Longin domain allowed it to extensively integrate into the endomembrane trafficking machinery in the earliest stages of building the eukaryotic cell.     

Protein trafficking in plant cells: Tools and markers

Eukaryotic cells consist of numerous membrane-bound organelles,which compartmentalize cellular materials to fulfil a variety of vital functions.In the post-genomic era,it is widely recognized that identification of the subcellular organelle localization and transport mechanisms of the encoded proteins are necessary for a fundamental understanding of their biological functions and the organization of cellular activity.Multiple experimental approaches are now available to determine the subcellular localizations and dynamics of proteins.In this review,we provide an overview of the current methods and organelle markers for protein subcellular localization and trafficking studies in plants,with a focus on the organelles of the endomembrane system.We also discuss the limitations of each method in terms of protein colocalization studies.     

Early steps in plastid evolution: current ideas and controversies

Some nuclear‐encoded proteins are imported into higher plant plastids via the endomembrane (EM) system. Compared with multi‐protein Toc and Tic translocons required for most plastid protein import, the relatively uncomplicated nature of EM trafficking led to suggestions that it was the original transport mechanism for nuclear‐encoded endosymbiont proteins, and critical for the early stages of plastid evolution. Its apparent simplicity disappears, however, when EM transport is considered in light of selective constraints likely encountered during the conversion of stable endosymbionts into fully integrated organelles. From this perspective it is more parsimonious to presume the early evolution of post‐translational protein import via simpler, ancestral forms of modern Toc and Tic plastid translocons, with EM trafficking arising later to accommodate glycosylation and/or protein targeting to multiple cellular locations. This hypothesis is supported by both empirical and comparative data, and is consistent with the relative paucity of EM‐based transport to modern primary plastids.     

Insights into the role of the membranes in Rab GTPase regulation

Rab GTPases are molecular switches with essential roles in mediating vesicular trafficking and establishing organelle identity. The conversion from the inactive, cytosolic to the membrane-bound, active species and back is tightly controlled by regulatory proteins. Recently, the roles of membrane properties and lipid composition of different target organelles in determining the activity state of Rabs have come to light. The investigation of several Rab guanine nucleotide exchange factors (GEFs) has revealed principles of how the recruitment via lipid interactions and the spatial confinement on the membrane surface contribute to spatiotemporal specificity in the Rab GTPase network. This paints an intricate picture of the control mechanisms in Rab activation and highlights the importance of the membrane lipid code in the organization of the endomembrane system.     

Endomembranes and vesicle trafficking

Over the past year extensive analyses of the accumulated data on the structural and functional organisation of the endomembrane system and vesicular trafficking in higher plants have shown it to be far more complex than previously anticipated. The availability of molecular tools combined with new opportunities to visualise endomembrane dynamics in vivo will allow better understanding of the fundamental processes underlying the complexity of endomembrane behaviour and vesicular trafficking.     

Brefeldin A promotes hydrolysis of sphingomyelin.

The hydrolysis of sphingomyelin (SM) is a key reaction in the "sphingomyelin cycle," which plays a role in the regulation of cell proliferation and differentiation (Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) J. Biol. Chem. 264, 19076-19080). SM is produced from endoplasmic reticulum-derived ceramide and is delivered to organelle membranes in a regulated manner, presumably through the same endomembrane trafficking system used for sorting and delivery of proteins. Since brefeldin A (BFA) interferes with this endomembrane trafficking system and thus alters normal membrane and organelle distribution, we investigated the effect of BFA on SM levels in HL-60 leukemia cells. BFA caused a dose-dependent decrease of 20-25% in cellular SM levels, with effects observed at concentrations of BFA as low as 0.10 microgram/ml. BFA effects on SM levels were noted as early as 5 min and were maximal by 20 min, with no further SM hydrolysis observed up to 60 min following treatment with BFA, suggesting the presence of a fixed SM-sensitive pool. BFA did not cause SM hydrolysis at 16 degrees C, a temperature that inhibits the effects of BFA on endomembrane mixing. The very early effects and temperature dependence of BFA-induced SM hydrolysis suggest that the mechanism of hydrolysis may be closely related to endomembrane mixing. These studies are beginning to define important interrelationships between membrane trafficking and topology, SM metabolism, and cell regulation.     

A proteomic approach to the bilirubin‐induced toxicity in neuronal cells reveals a protective function of DJ‐1 protein

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long‐term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB‐induced neuronal toxicity, we used the human neuroblastoma cell line SH‐SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ‐1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin‐induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.     

Impact of sphingolipids on protein membrane trafficking

Membrane trafficking is essential to maintain the spatiotemporal control of protein and lipid distribution within membrane systems of eukaryotic cells. To achieve their functional destination proteins are sorted and transported into lipid carriers that construct the secretory and endocytic pathways. It is an emerging theme that lipid diversity might exist in part to ensure the homeostasis of these pathways. Sphingolipids, a chemical diverse type of lipids with special physicochemical characteristics have been implicated in the selective transport of proteins. In this review, we will discuss current knowledge about how sphingolipids modulate protein trafficking through the endomembrane systems to guarantee that proteins reach their functional destination and the proposed underlying mechanisms.     

Molecular interplay of autophagy and endocytosis in human health and diseases

Autophagy, an evolutionarily conserved process for maintaining the physio‐metabolic equilibrium of cells, shares many common effector proteins with endocytosis. For example, tethering proteins involved in fusion like Ras‐like GTPases (Rabs), soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs), lysosomal‐associated membrane protein (LAMP), and endosomal sorting complex required for transport (ESCRT) have a dual role in endocytosis and autophagy, and the trafficking routes of these processes converge at lysosomes. These common effectors indicate an association between budding and fusion of membrane‐bound vesicles that may have a substantial role in autophagic lysosome reformation, by sensing cellular stress levels. Therefore, autophagy–endocytosis crosstalk may be significant and implicates a novel endocytic regulatory pathway of autophagy. Moreover, endocytosis has a pivotal role in the intake of signalling molecules, which in turn activates cascades that can result in pathophysiological conditions. This review discusses the basic mechanisms of this crosstalk and its implications in order to identify potential novel therapeutic targets for various human diseases.     

Sortin2 enhances endocytic trafficking towards the vacuole in Saccharomyces cerevisiae

Background

A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae.

Results

A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked.

Conclusions

Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0032-9) contains supplementary material, which is available to authorized users.     

Traffic jams affect plant development and signal transduction

Analysis of the Arabidopsis thaliana endomembrane system has shown that plant cell viability depends on a properly functioning vacuole and intact vesicular trafficking. The endomembrane system is also essential for various aspects of plant development and signal transduction. In this review, we discuss examples of these newly discovered roles for the endomembrane system in plants, and new experimental approaches and technologies that are based on high-throughput screens, which combine chemical genetics and automated confocal microscopy.     

TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy

The RabGAP protein TBC1D5 controls cellular endomembrane trafficking processes and binds the retromer subunit VPS29 and the ubiquitin‐like protein ATG8 (LC3). Here, we describe that TBC1D5 also associates with ATG9 and the active ULK1 complex during autophagy. Moreover, ATG9 and TBC1D5 interact with clathrin and the AP2 complex. Depletion of TBC1D5 leads to missorting of ATG9 to late endosomes upon activation of autophagy, whereas inhibition of clathrin‐mediated endocytosis or AP2 depletion alters ATG9 trafficking and its association with TBC1D5. Taken together, our data show that TBC1D5 and the AP2 complex are important novel regulators of the rerouting of ATG9‐containing vesicular carriers toward sites of autophagosome formation.     

Cellular distribution of secretory pathway markers in the haploid synergid cells of Arabidopsis thaliana

In flowering plants, cell–cell communication plays a key role in reproductive success, as both pollination and fertilization require pathways that regulate interactions between many different cell types. Some of the most critical of these interactions are those between the pollen tube (PT) and the embryo sac, which ensure the delivery of sperm cells required for double fertilization. Synergid cells function to attract the PT through secretion of small peptides and in PT reception via membrane‐bound proteins associated with the endomembrane system and the cell surface. While many synergid‐expressed components regulating PT attraction and reception have been identified, few tools exist to study the localization of membrane‐bound proteins and the components of the endomembrane system in this cell type. In this study, we describe the localization and distribution of seven fluorescent markers that labelled components of the secretory pathway in synergid cells of Arabidopsis thaliana. These markers were used in co‐localization experiments to investigate the subcellular distribution of the two PT reception components LORELEI, a GPI‐anchored surface protein, and NORTIA, a MILDEW RESISTANCE LOCUS O protein, both found within the endomembrane system of the synergid cell. These secretory markers are useful tools for both reproductive and cell biologists, enabling the analysis of membrane‐associated trafficking within a haploid cell actively involved in polar transport.