The ins and outs of yeast vacuole trafficking

Summary Vacuoles are ubiquitous organelles in the fungal and plant kingdoms. They serve a variety of functions and are important for cell homeostasis. A constant turnover of proteins and membranes makes vacuoles dynamic organelles. Various transport pathways share the vacuole as their joint destination. The trafficking pathways are regulated independently. In yeast cells many components of the protein and membrane transport machinery are known. Recent years have seen much progress in our understanding of the protein-sorting pathways and the biogenesis of this organelle. Improvements of our understanding of the vesicular transport pathways and vacuolar membrane fusion are reviewed.     

Molecular mechanisms of autophagic peroxisome degradation in yeasts

Autophagy, Cvt pathway and pexophagy belong to membrane transport routes, which are able to enwrap into double-membrane vesicles and deliver to the vacuole various cytosolic material, including organelles. Pexophagy is a selective pathway of vacuolar degradation of redundant peroxisomes and can be induced by certain changes of carbon sources in yeasts. Here we review the most general molecular mechanisms of autophagic transport routes with a special emphasis on their features and functions in the yeast peroxisome degradation. Special attention has been also paid to differences in functioning of the basic autophagic machinery during micro- and macroautophagic peroxisome degradation in methylotrophic yeasts. The requirements of autophagic pathways for the sources of membrane for transport vesicle formation are also analyzed. Finally, we point to the gaps in our understanding of peroxisome degradation, which should be filled for complete integration of pexophagy into the network of autophagic transport routes to the vacuole in yeast.     

Intracellular trafficking of sphingolipids: Relationship to biosynthesis

The intracellular routes of sphingolipid trafficking are related to the compartmentalized nature of sphingolipid metabolism, with synthesis beginning in the endoplasmic reticulum, continuing in the Golgi apparatus, and degradation occurring mainly in lysosomes. Whereas bulk sphingolipid transport between subcellular organelles occurs primarily via vesicle-mediated pathways, evidence is accumulating that sphingolipids are found in subcellular organelles that are not connected to each other by vesicular flow, implying additional trafficking routes. After discussing how sphingolipids are transported through the secretory pathway, I will review evidence for sphingolipid metabolism in organelles such as the mitochondria, and then discuss how this impacts upon our current understanding of the regulation of intracellular sphingolipid transport.     

Intracellular trafficking of sphingolipids: relationship to biosynthesis

The intracellular routes of sphingolipid trafficking are related to the compartmentalized nature of sphingolipid metabolism, with synthesis beginning in the endoplasmic reticulum, continuing in the Golgi apparatus, and degradation occurring mainly in lysosomes. Whereas bulk sphingolipid transport between subcellular organelles occurs primarily via vesicle-mediated pathways, evidence is accumulating that sphingolipids are found in subcellular organelles that are not connected to each other by vesicular flow, implying additional trafficking routes. After discussing how sphingolipids are transported through the secretory pathway, I will review evidence for sphingolipid metabolism in organelles such as the mitochondria, and then discuss how this impacts upon our current understanding of the regulation of intracellular sphingolipid transport.     

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, integrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations.     

Specialized ER membrane domains for lipid metabolism and transport

The endoplasmic reticulum (ER) is a highly organized organelle that performs vital functions including de novo membrane lipid synthesis and transport. Accordingly, numerous lipid biosynthesis enzymes are localized in the ER membrane. However, it is now evident that lipid metabolism is sub-compartmentalized within the ER and that lipid biosynthetic enzymes engage with lipid transfer proteins (LTPs) to rapidly shuttle newly synthesized lipids from the ER to other organelles. As such, intimate relationships between lipid metabolism and lipid transfer pathways exist within the ER network. Notably, certain LTPs enhance the activities of lipid metabolizing enzymes; likewise, lipid metabolism can ensure the specificity of LTP transfer/exchange reactions. Yet, our understanding of these mutual relationships is still emerging. Here, we highlight past and recent key findings on specialized ER membrane domains involved in efficient lipid metabolism and transport and consider unresolved issues in the field.     

Spatial and Temporal Characteristics of Normal and Perturbed Vesicle Transport

Efficient intracellular transport is essential for healthy cellular function and structural integrity, and problems in this pathway can lead to neuronal cell death and disease. To spatially and temporally evaluate how transport defects are initiated, we adapted a primary neuronal culture system from Drosophila larval brains to visualize the movement dynamics of several cargos/organelles along a 90 micron axonal neurite over time. All six vesicles/organelles imaged showed robust bi-directional motility at both day 1 and day 2. Reduction of motor proteins decreased the movement of vesicles/organelles with increased numbers of neurite blocks. Neuronal growth was also perturbed with reduction of motor proteins. Strikingly, we found that all blockages were not fixed, permanent blocks that impeded transport of vesicles as previously thought, but that some blocks were dynamic clusters of vesicles that resolved over time. Taken together, our findings suggest that non-resolving blocks may likely initiate deleterious pathways leading to death and degeneration, while resolving blocks may be benign. Therefore evaluating the spatial and temporal characteristics of vesicle transport has important implications for our understanding of how transport defects can affect other pathways to initiate death and degeneration.     

Molecular landmarks along the axonal route: axonal transport in health and disease

Axonal transport of organelles has emerged as a key process in the regulation of neuronal differentiation and survival. Several components of this specialised transport machinery, their regulators and vesicular cargoes are mutated or altered in many neurodegenerative conditions. The molecular characterisation of these mechanisms has furthered our understanding of neuronal homeostasis, providing insights into the spatio-temporal control of membrane traffic and signalling in neurons with a precision not achievable in other cellular systems. Here, we summarise the recent advances in the field of axonal trafficking of different organelles, and the essential role of motor and adaptor proteins in this process.     

Getting ready for building: signaling and autophagosome biogenesis

Autophagy is the main cellular catabolic process responsible for degrading organelles and large protein aggregates. It is initiated by the formation of a unique membrane structure, the phagophore, which engulfs part of the cytoplasm and forms a double‐membrane vesicle termed the autophagosome. Fusion of the outer autophagosomal membrane with the lysosome and degradation of the inner membrane contents complete the process. The extent of autophagy must be tightly regulated to avoid destruction of proteins and organelles essential for cell survival. Autophagic activity is thus regulated by external and internal cues, which initiate the formation of well‐defined autophagy‐related protein complexes that mediate autophagosome formation and selective cargo recruitment into these organelles. Autophagosome formation and the signaling pathways that regulate it have recently attracted substantial attention. In this review, we analyze the different signaling pathways that regulate autophagy and discuss recent progress in our understanding of autophagosome biogenesis.     

Organelle-localized potassium transport systems in plants

Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K⁺ transport systems allowing K⁺ to move across the membrane. K⁺ transport systems in plant organelles act coordinately with the plasma membrane intrinsic K⁺ transport systems to maintain cytosolic K⁺ concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K⁺ channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K⁺ homeostasis of the cytoplasm. The initial electrophysiological measurements of K⁺ transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K⁺ transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K⁺ transport system has been isolated from cyanobacteria, which may add to our understanding of K⁺ flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K⁺ transport proteins.     

Protein translocation across membranes.

Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.     

Glycerolipid transfer for the building of membranes in plant cells

Membranes of plant organelles have specific glycerolipid compositions. Selective distribution of lipids at the levels of subcellular organelles, membrane leaflets and membrane domains reflects a complex and finely tuned lipid homeostasis. Glycerolipid neosynthesis occurs mainly in plastid envelope and endoplasmic reticulum membranes. Since most lipids are not only present in the membranes where they are synthesized, one cannot explain membrane specific lipid distribution by metabolic processes confined in each membrane compartment. In this review, we present our current understanding of glycerolipid trafficking in plant cells. We examine the potential mechanisms involved in lipid transport inside bilayers and from one membrane to another. We survey lipid transfers going through vesicular membrane flow and those dependent on lipid transfer proteins at membrane contact sites. By introducing recently described membrane lipid reorganization during phosphate deprivation and recent developments issued from mutant analyses, we detail the specific lipid transfers towards or outwards the chloroplast envelope.     

Cellular magnesium homeostasis

Magnesium, the second most abundant cellular cation after potassium, is essential to regulate numerous cellular functions and enzymes, including ion channels, metabolic cycles, and signaling pathways, as attested by more than 1000 entries in the literature. Despite significant recent progress, however, our understanding of how cells regulate Mg²⁺ homeostasis and transport still remains incomplete. For example, the occurrence of major fluxes of Mg²⁺ in either direction across the plasma membrane of mammalian cells following metabolic or hormonal stimuli has been extensively documented. Yet, the mechanisms ultimately responsible for magnesium extrusion across the cell membrane have not been cloned. Even less is known about the regulation in cellular organelles. The present review is aimed at providing the reader with a comprehensive and up-to-date understanding of the mechanisms enacted by eukaryotic cells to regulate cellular Mg²⁺ homeostasis and how these mechanisms are altered under specific pathological conditions.     

Endoplasmic reticulum–plasma membrane contacts: Principals of phosphoinositide and calcium signaling

The endoplasmic reticulum (ER) forms an extensive network of membrane contact sites with intra-cellular organelles and the plasma membrane (PM). Interorganelle contacts have vital roles in membrane lipid and ion dynamics. In particular, ER–PM contacts are integral to numerous inter-cellular and intra-cellular signaling pathways including phosphoinositide lipid and calcium signaling, mechanotransduction, metabolic regulation, and cell stress responses. Accordingly, ER–PM contacts serve important signaling functions in excitable cells including neurons and muscle and endocrine cells. This review highlights recent advances in our understanding of the vital roles for ER–PM contacts in phosphoinositide and calcium signaling and how signaling pathways in turn regulate proteins that form and function at ER–PM contacts.     

RAB GTPases and SNAREs at the trans-Golgi network in plants

Membrane traffic is a fundamental cellular system to exchange proteins and membrane lipids among single membrane-bound organelles or between an organelle and the plasma membrane in order to keep integrity of the endomembrane system. RAB GTPases and SNARE proteins, the key regulators of membrane traffic, are conserved broadly among eukaryotic species. However, genome-wide analyses showed that organization of RABs and SNAREs that regulate the post-Golgi transport pathways is greatly diversified in plants compared to other model eukaryotes. Furthermore, some organelles acquired unique properties in plant lineages. Like in other eukaryotic systems, the trans-Golgi network of plants coordinates secretion and vacuolar transport; however, uniquely in plants, it also acts as a platform for endocytic transport and recycling. In this review, we focus on RAB GTPases and SNAREs that function at the TGN, and summarize how these regulators perform to control different transport pathways at the plant TGN. We also highlight the current knowledge of RABs and SNAREs’ role in regulation of plant development and plant responses to environmental stimuli.

     

Cholesterol trafficking in the secretory and endocytic systems

Cells maintain a cholesterol gradient across the secretory system, with the lowest concentrations in the endoplasmic reticulum (ER) and the highest in the plasma membrane (PM). Cholesterol is also heterogeneously distributed in the endocytic pathway. Little is known about how this heterogeneous distribution of cholesterol is maintained despite continuous vesicular traffic between organelles. Both the modulation of the cholesterol content of transport vesicles and the non-vesicular transport of cholesterol between organelles are likely to contribute. This review summarizes what is known about the pathways and mechanisms of intracellular sterol trafficking.     

Transporters for uptake and allocation of organic nitrogen compounds in plants

Nitrogen is an essential macronutrient for plant growth. Following uptake from the soil or assimilation within the plant, organic nitrogen compounds are transported between organelles, from cell to cell and over long distances in support of plant metabolism and development. These translocation processes require the function of integral membrane transporters. The review summarizes our current understanding of the molecular mechanisms of organic nitrogen transport processes, with a focus on amino acid, ureide and peptide transporters.     

Targeting motifs in GLUT4

The trafficking of the insulin-sensitive glucose transporter, GLUT4, is the paradigm of how cells control the movement of membrane proteins through intricate pathways of transport in response to external stimuli, and how, by doing so, regulate their function. The GLUT4 intracellularly sequestered in resting adipocytes and muscle cells becomes exposed on their surface in response to an increase in insulin levels and muscle contraction, where it facilitates glucose uptake. Ceasing of the stimuli is followed by endocytosis of the GLUT4 molecules exposed on the plasma membrane and their recycling to the original stores, where they are retained. This review discusses current understanding of the organelles that host GLUT4 and the motifs that mediate its trafficking.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

Targeting motifs in GLUT4 (review).

The trafficking of the insulin-sensitive glucose transporter, GLUT4, is the paradigm of how cells control the movement of membrane proteins through intricate pathways of transport in response to external stimuli, and how, by doing so, regulate their function. The GLUT4 intracellularly sequestered in resting adipocytes and muscle cells becomes exposed on their surface in response to an increase in insulin levels and muscle contraction, where it facilitates glucose uptake. Ceasing of the stimuli is followed by endocytosis of the GLUT4 molecules exposed on the plasma membrane and their recycling to the original stores, where they are retained. This review discusses current understanding of the organelles that host GLUT4 and the motifs that mediate its trafficking.