Reciprocal Regulation of Endocytosis and Metabolism

The cellular uptake of many nutrients and micronutrients governs both their cellular availability and their systemic homeostasis. The cellular rate of nutrient or ion uptake (e.g., glucose, Fe³⁺, K⁺) or efflux (e.g., Na⁺) is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these proteins on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis.Interactions between the cell and its environment obligatorily involve events at the plasma membrane. Cell-surface proteins mediate nutrient uptake, product release, and the sensing of environmental changes, including signals from other cells. Appropriate sensing and response to extracellular cues is essential for the individual cell’s survival and for the coordinated cellular behavior in multicellular organisms. Accordingly, maintenance and dynamics of membrane proteins are fundamental mechanisms of cellular homeostasis and survival.Most plasma membrane proteins are in defined equilibria with intracellular endosomal compartments, such that the amount of a given protein at the plasma membrane is determined by the balance of its endocytosis and its recycling back to the cell surface from endosomes and other intracellular compartments (Fig. 1). Changes in the kinetics of membrane protein traffic acutely affect the levels of individual proteins at the cell surface and thereby impact how cells intake nutrients, sense the environment, and respond to external cues.Open in a separate windowFigure 1.Dynamic regulation of the cell-surface content of membrane proteins. Integral membrane proteins found at the cell surface are dynamically localized to the plasma membrane. The amount of any of these proteins at the cell surface is the result of the balance of exocytosis or recycling of vesicles containing that protein from intracellular membrane compartments and the endocytosis of the protein from the cell surface. Regulation of either the rate of exocytosis or endocytosis results in alteration of the cell-surface content of a given protein.Selective molecular mechanisms trigger traffic of plasma membrane proteins through endomembranes. Among them, ubiquitination and phosphorylation stand out as they can directly target the cargo proteins. Ubiquitination is the covalent attachment of the 76-amino acid polypeptide ubiquitin to the ε-amino group of specific lysine residues (reviewed by Miranda and Sorkin 2007; and see also Piper et al. 2014). Ubiquitination of cell-surface proteins is the principal mechanism of control of endocytosis in yeast (MacGurn et al. 2012), whereas in mammals, additional molecular mechanisms regulate the endocytosis of cell-surface proteins, including alterations in conformation that impact interaction with other proteins, and as mentioned, phosphorylation. Each of these modifications can either enhance or reduce the rates internalization, recycling, or degradation of specific proteins, highlighting the complexity of the regulation of endomembrane traffic. The intricate mechanisms that underlie the reciprocal regulation of endocytosis and metabolism are beginning to be understood. Here we discuss the endocytosis mechanisms in the regulation of cellular intake or efflux of iron, cholesterol, Na⁺, and glucose, and in the regulation of receptor signaling relevant to metabolism.     

Rab GTPases regulating receptor trafficking at the late endosome–lysosome membranes

Lysosomes serve key degradative functions for the turnover of membrane lipids and protein components. Its biogenesis is principally dependent on exocytic traffic from the late endosome via the trans‐Golgi network, and it also receives cargo to be degraded from the endocytic pathway. Membrane trafficking to the late endosome–lysosome is tightly regulated to maintain the amplitude of signalling events and cellular homeostasis. Key coordinators of lysosomal traffic include members of the Rab small GTPase family. Amongst these, Rab7, Rab9 and the more recently studied Rab22B/31 have all been reported to regulate membrane trafficking processed at the late endosome–lysosome system. We discuss what is known about the roles of these Rab proteins and their interacting partners on the regulation of traffic of important receptor proteins such as the epidermal growth factor receptor (EGFR) and the mannose 6‐phosphate receptor (M6PR), in association with the late endosome–lysosome system. Better knowledge of EGFR and M6PR traffic in this regard may aid in understanding the pathological processes, such as oncogenic transformations associated with these receptors. Copyright © 2012 John Wiley & Sons, Ltd.     

Lysosomal Membrane Proteins and Their Central Role in Physiology

The lysosomal membrane was thought for a long time to primarily act as a physical barrier separating the luminal acidic milieu from the cytoplasmic environment. Meanwhile, it has been realized that unique lysosomal membranes play essential roles in a number of cellular events ranging from phagocytosis, autophagy, cell death, virus infection to membrane repair. This review provides an overview about the most interesting emerging functions of lysosomal membrane proteins and how they contribute to health and disease. Their importance is exemplified by their role in acidification, transport of metabolites and ions across the membrane, intracellular transport of hydrolases and the regulation of membrane fusion events. Studies in patient cells, non‐mammalian model organisms and knockout mice contributed to our understanding of how the different lysosomal membrane proteins affect cellular homeostasis, developmental processes as well as tissue functions. Because these proteins are central for the biogenesis of this compartment they are also considered as attractive targets to modulate the lysosomal machinery in cases where impaired lysosomal degradation leads to cellular pathologies. We are only beginning to understand the complex composition and function of these proteins which are tightly linked to processes occurring throughout the endocytic and biosynthetic pathways.      

From the inside out: Ion fluxes at the centre of endocytic traffic

Endocytic traffic is a complex and elegant operation involving cargo sorting, membrane budding and tubulation, generation of force, and the formation of organellar contacts. The role of specific proteins and lipids in these processes has been studied extensively. By comparison, precious little is understood about the contribution of the endocytic fluid to these events, despite much evidence that alteration of the contents can severely affect membrane traffic along the endocytic pathway. In particular, it has long been appreciated that dissipation of ionic gradients arrests endosome-to-lysosome maturation. How cells sense inorganic ions and transmit this information have remained largely enigmatic. Herein, we review the experimental findings that reveal an intimate association between luminal ions, their transport, and endocytic traffic. We then discuss the ionic sensors and the mechanisms proposed to convert ion concentrations into protein-based trafficking events, highlighting the current paucity of convincing explanations.     

Cellular functions and intrinsic attributes of the ATP‐binding Eps15 homology domain‐containing proteins

Several cellular processes rely on a cohort of dedicated proteins that manage tubulation, fission, and fusion of membranes. A notably large number of them belong to the dynamin superfamily of proteins. Among them is the evolutionarily conserved group of ATP‐binding Eps15‐homology domain‐containing proteins (EHDs). In the two decades since their discovery, EHDs have been linked to a range of cellular processes that require remodeling or maintenance of specific membrane shapes such as during endocytic recycling, caveolar biogenesis, ciliogenesis, formation of T‐tubules in skeletal muscles, and membrane resealing after rupture. Recent work has shed light on their structure and the unique attributes they possess in linking ATP hydrolysis to membrane remodeling. This review summarizes some of these recent developments and reconciles intrinsic protein functions to their cellular roles.     

Investigation of penetratin peptides. Part 2. In vitro uptake of penetratin and two of its derivatives

As endocytic uptake of the Antennapedia homeodomain‐derived penetratin peptide (RQIKIWFQNRRMKWKK) is finally being revealed, some of the early views about penetratin need to be reconsidered. Endocytic uptake seems to contradict the indispensability of tryptophans and also the minimum length of 16 amino acid residues for efficient internalization. To revise the membrane translocation of penetratin, two penetratin analogs were designed and synthesized: a peptide in which tryptophans were replaced by phenylalanines (Phe^{6, 14}‐penetratin, RQIKI F FQNRRMK F KK) and a shortened analog (dodeca‐penetratin, RQIKIWF‐R‐KWKK) made up of only 12 residues. The peptides were fluorescently labeled and applied to live, unfixed cells from various lines. Cellular uptake was analysed by confocal microscopy and flow cytometry. Low temperature or ATP‐depletion blocked the intracellular entry of all three penetratin peptides. A decrease in membrane fluidity or cholesterol depletion with methyl‐β‐cyclodextrin greatly inhibited peptide uptake, showing the involvement of cholesterol‐rich lipid rafts in internalization. Exogenous heparan sulfate also diminished the internalization of penetratin and its derivatives, reflecting the paramount importance of electrostatic interactions with polyanionic cell‐surface proteoglycans. The beneficial presence of tryptophans is supported by observations on the decreased cellular uptake of Phe^{6, 14}‐penetratin. The maintained translocational efficiency of dodeca‐penetratin demonstrates that a thorough understanding of penetratin internalization can yield new penetratin analogs with unaltered translocational abilities. This study provides evidence on the energy‐dependent and lipid raft‐mediated endocytic uptake of penetratin and highlights the necessity of revealing those pathways that cationic cell‐penetrating peptides employ to enter live cells. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

CLIP‐170 spatially modulates receptor tyrosine kinase recycling to coordinate cell migration

Endocytic sorting of activated receptor tyrosine kinases (RTKs), alternating between recycling and degradative processes, controls signal duration, location and surface complement of RTKs. The microtubule (MT) plus‐end tracking proteins (+TIPs) play essential roles in various cellular activities including translocation of intracellular cargo. However, mechanisms through which RTKs recycle back to the plasma membrane following internalization in response to ligand remain poorly understood. We report that net outward‐directed movement of endocytic vesicles containing the hepatocyte growth factor (HGF) Met RTK, requires recruitment of the +TIP, CLIP‐170, as well as the association of CLIP‐170 to MT plus‐ends. In response to HGF, entry of Met into Rab4‐positive endosomes results in Golgi‐localized γ‐ear‐containing Arf‐binding protein 3 (GGA3) and CLIP‐170 recruitment to an activated Met RTK complex. We conclude that CLIP‐170 co‐ordinates the recycling and the transport of Met‐positive endocytic vesicles to plus‐ends of MTs towards the cell cortex, including the plasma membrane and the lamellipodia, thereby promoting cell migration.      

Staying in touch with the endocytic network: The importance of contacts for cholesterol transport

Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter‐dependent vesicular and non‐vesicular transport mechanisms. The last decade has seen a surge in interest in non‐vesicular transport and inter‐organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co‐ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non‐vesicular transport mechanisms and relationship with neurodegenerative disease.     

Chemistry‐based protein modification strategy for endocytic pathway analysis

Background information. The integrated analysis of intracellular trafficking pathways is one of the current challenges in the field of cell biology, and functional proteomics has become a powerful technique for the large‐scale identification of proteins or lipids and the elucidation of biological processes in their natural contexts. For this, new dynamic strategies must be devised to trace proteins that follow a specific pathway such that their initial and final destinations can be detected by automated means. Results. Here, we report a novel vectorial strategy for trafficking pathway analysis. This strategy is based on a chemical modification of plasma membrane proteins with a bSuPeR (biotinylated sulfation site peptide reagent) and metabolic labelling in the Golgi apparatus, such that plasma membrane proteins that traffic via the retrograde route become detectable in complex mixtures. Efficient synthesis schemes are presented for tailor‐made chemical tools that are then applied to the step‐by‐step validation of the strategy, using a known retrograde cargo protein: the STxB (Shiga toxin B‐subunit). bSuPeR modification at the plasma membrane does not affect STxB transport to the Golgi apparatus, where the protein is metabolically labelled, allowing its detection in cell lysates. Conclusions. Our vectorial concept proposes a new chemical approach for traffic‐based profiling of proteins that may prove to be applicable to the analysis of diverse endocytic pathways.     

Tying trafficking to fusion and fission at the mighty mitochondria

The mitochondrion is a unique organelle that serves as the main site of ATP generation needed for energy in the cell. However, mitochondria also play essential roles in cell death through apoptosis and necrosis, as well as a variety of crucial functions related to stress regulation, autophagy, lipid synthesis and calcium storage. There is a growing appreciation that mitochondrial function is regulated by the dynamics of its membrane fusion and fission; longer, fused mitochondria are optimal for ATP generation, whereas fission of mitochondria facilitates mitophagy and cell division. Despite the significance of mitochondrial homeostasis for such crucial cellular events, the intricate regulation of mitochondrial fusion and fission is only partially understood. Until very recently, only a single mitochondrial fission protein had been identified. Moreover, only now have researchers turned to address the upstream machinery that regulates mitochondrial fusion and fission proteins. Herein, we review the known GTPases involved in mitochondrial fusion and fission, but also highlight recent studies that address the mechanisms by which these GTPases are regulated. In particular, we draw attention to a substantial new body of literature linking endocytic regulatory proteins, such as the retromer VPS35 cargo selection complex subunit, to mitochondrial homeostasis. These recent studies suggest that relationships and cross‐regulation between endocytic and mitochondrial pathways may be more widespread than previously assumed.      

Phosphatidylinositol 3‐phosphates—at the interface between cell signalling and membrane traffic

Phosphoinositides (PIs) form a minor class of phospholipids with crucial functions in cell physiology, ranging from cell signalling and motility to a role as signposts of compartmental membrane identity. Phosphatidylinositol 3‐phosphates are present at the plasma membrane and within the endolysosomal system, where they serve as key regulators of both cell signalling and of intracellular membrane traffic. Here, we provide an overview of the metabolic pathways that regulate cellular synthesis of PI 3‐phosphates at distinct intracellular sites and discuss the mechanisms by which these lipids regulate cell signalling and membrane traffic. Finally, we provide a framework for how PI 3‐phosphate metabolism is integrated into the cellular network.     

Quantitative visualization of endocytic trafficking through photoactivation of fluorescent proteins

Endocytic trafficking controls the density of molecules at the plasma membrane and by doing so, the cell surface profile, which in turn determines how cells interact with their environment. A full apprehension of any cellular process necessitates understanding how proteins associated with the plasma membrane are endocytosed, how they are sorted after internalization, and if and how they are recycled to the plasma membrane. To date, it is still difficult to experimentally gain access to this information, even more to do it in a quantitative way. Here we present a toolset based on photoactivation of fluorescent proteins that enabled us to generate quantitative information on endocytosis, incorporation into sorting and recycling endosomes, delivery from endosomes to the plasma membrane, and on the type of vesicles performing intracellular transport. We illustrate these approaches by revealing striking differences in the endocytic trafficking of T-cell receptor and CD4, which bind to the same molecule at the surface of antigen-presenting cells during T-cell activation.     

Endosome maturation factors Rabenosyn‐5/VPS45 and caveolin‐1 regulate ciliary membrane and polycystin‐2 homeostasis

Primary cilium structure and function relies on control of ciliary membrane homeostasis, regulated by membrane trafficking processes that deliver and retrieve ciliary components at the periciliary membrane. However, the molecular mechanisms controlling ciliary membrane establishment and maintenance, especially in relation to endocytosis, remain poorly understood. Here, using Caenorhabditis elegans, we describe closely linked functions for early endosome (EE) maturation factors RABS‐5 (Rabenosyn‐5) and VPS‐45 (VPS45) in regulating cilium length and morphology, ciliary and periciliary membrane volume, and ciliary signalling‐related sensory behaviour. We demonstrate that RABS‐5 and VPS‐45 control periciliary vesicle number and levels of select EE/endocytic markers (WDFY‐2, CAV‐1) and the ciliopathy membrane receptor PKD‐2 (polycystin‐2). Moreover, we show that CAV‐1 (caveolin‐1) also controls PKD‐2 ciliary levels and associated sensory behaviour. These data link RABS‐5 and VPS‐45 ciliary functions to the processing of periciliary‐derived endocytic vesicles and regulation of ciliary membrane homeostasis. Our findings also provide insight into the regulation of PKD‐2 ciliary levels via integrated endosomal sorting and CAV‐1‐mediated endocytosis.     

Cadherin tales: Regulation of cadherin function by endocytic membrane trafficking

Cadherins are the primary adhesion molecules in adherens junctions and desmosomes and play essential roles in embryonic development. Although significant progress has been made in understanding cadherin structure and function, we lack a clear vision of how cells confer plasticity upon adhesive junctions to allow for cellular rearrangements during development, wound healing and metastasis. Endocytic membrane trafficking has emerged as a fundamental mechanism by which cells confer a dynamic state to adhesive junctions. Recent studies indicate that the juxtamembrane domain of classical cadherins contains multiple endocytic motifs, or “switches,” that can be used by cellular membrane trafficking machinery to regulate adhesion. The cadherin‐binding protein p120‐catenin (p120) appears to be the master regulator of access to these switches, thereby controlling cadherin endocytosis and turnover. This review focuses on p120 and other cadherin‐binding proteins, ubiquitin ligases, and growth factors as key modulators of cadherin membrane trafficking.      

Endocytic membrane trafficking in the control of centrosome function

Until recently, endocytic trafficking and its regulators were thought to function almost exclusively on membrane-bound organelles and/or vesicles containing a lipid bilayer. Recent studies have demonstrated that endocytic regulatory proteins play much wider roles in trafficking regulation and influence a variety of nonendocytic pathways, including trafficking to/from mitochondria and peroxisomes. Moreover, new studies also suggest that endocytic regulators also control trafficking to and from cellular organelles that lack membranes, such as the centrosome. Although endocytic membrane trafficking (EMT) clearly impacts pathways downstream of the centrosome, such as ciliogenesis (including transport to and from cilia), mitotic spindle formation, and cytokinesis, relatively few studies have focused on the growing role for EMT more directly on centrosome biogenesis, maintenance and control throughout cell cycle, and centrosome duplication. Indeed, a growing number of endocytic regulatory proteins have been implicated in centrosome regulation, including various Rab proteins (among them Rab11) and the leucine-rich repeat kinase 2. In this review, we will examine the relationship between centrosomes and EMT, focusing primarily on how EMT directly influences the centrosome.     

Retrograde trafficking and plasma membrane recycling pathways of the budding yeast Saccharomyces cerevisiae

The endosomal system functions as a network of protein and lipid sorting stations that receives molecules from endocytic and secretory pathways and directs them to the lysosome for degradation, or exports them from the endosome via retrograde trafficking or plasma membrane recycling pathways. Retrograde trafficking pathways describe endosome‐to‐Golgi transport while plasma membrane recycling pathways describe trafficking routes that return endocytosed molecules to the plasma membrane. These pathways are crucial for lysosome biogenesis, nutrient acquisition and homeostasis and for the physiological functions of many types of specialized cells. Retrograde and recycling sorting machineries of eukaryotic cells were identified chiefly through genetic screens using the budding yeast Saccharomyces cerevisiae system and discovered to be highly conserved in structures and functions. In this review, we discuss advances regarding retrograde trafficking and recycling pathways, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways.