The human LAT1-4F2hc (SLC7A5-SLC3A2) transporter complex: Physiological and pathophysiological implications.

LAT1 and 4F2hc form a heterodimeric membrane protein complex, which functions as one of the best characterized amino acid transporters. Since LAT1-4F2hc is required for the efficient uptake of essential amino acids and hormones, it promotes cellular growth, in part, by stimulating mTORC1 (mechanistic target of rapamycin complex 1) signalling and by repressing the integrated stress response (ISR). Gain or loss of LAT1-4F2hc function is associated with cancer, diabetes, and immunological and neurological diseases. Hence, LAT1-4F2hc represents an attractive drug target for disease treatment. Specific targeting of LAT1-4F2hc will be facilitated by the increasingly detailed understanding of its molecular architecture, which provides important concepts for its function and regulation. Here, we summarize (i) structural insights that help to explain how LAT1 and 4F2hc assemble to transport amino acids across membranes, (ii) the role of LAT1-4F2hc in key metabolic signalling pathways, and (iii) how derailing these processes could contribute to diseases.

Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1.

Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.

Protein quality control at the Golgi.

The majority of the proteome in eukaryotic cells is targeted to organelles. To maintain protein homeostasis (proteostasis), distinct protein quality control (PQC) machineries operate on organelles, where they detect misfolded proteins, orphaned and mis-localized proteins and selectively target these proteins into different ubiquitin-dependent or -independent degradation pathways. Thereby, PQC prevents proteotoxic effects that would disrupt organelle integrity and cause cellular damage that leads to diseases. Here, we will discuss emerging mechanisms for PQC machineries at the Golgi apparatus, the central station for the sorting and the modification of proteins that traffic to the endo-lysosomal system, or along the secretory pathway to the PM and to the extracellular space. We will focus on Golgi PQC pathways that (1) retrieve misfolded and orphaned proteins from the Golgi back to the endoplasmic reticulum, (2) extract these proteins from Golgi membranes for proteasomal degradation, (3) or selectively target these proteins to lysosomes for degradation.

The α-arrestin family of ubiquitin ligase adaptors links metabolism with selective endocytosis.

The regulation of nutrient uptake into cells is important, as it allows to either increase biomass for cell growth or to preserve homoeostasis. A key strategy to adjust cellular nutrient uptake is the reconfiguration of the nutrient transporter repertoire at the plasma membrane by the addition of nutrient transporters through the secretory pathway and by their endocytic removal. In this review, we focus on the mechanisms that regulate selective nutrient transporter endocytosis, which is mediated by the α-arrestin protein family. In the budding yeast Saccharomyces cerevisiae, 14 different α-arrestins (also named arrestin-related trafficking adaptors, ARTs) function as adaptors for the ubiquitin ligase Rsp5. They instruct Rsp5 to ubiquitinate subsets of nutrient transporters to orchestrate their endocytosis. The ART proteins are under multilevel control of the major nutrient sensing systems, including amino acid sensing by the general amino acid control and target of rapamycin pathways, and energy sensing by 5'-adenosine-monophosphate-dependent kinase. The function of the six human α-arrestins is comparably under-characterised. Here, we summarise the current knowledge about the function, regulation and substrates of yeast ARTs and human α-arrestins, and highlight emerging communalities and general principles.

SATB2-LEMD2 interaction links nuclear shape plasticity to regulation of cognition-related genes.

SATB2 is a schizophrenia risk gene and is genetically associated with human intelligence. How it affects cognition at molecular level is currently unknown. Here, we show that interactions between SATB2, a chromosomal scaffolding protein, and the inner nuclear membrane protein LEMD2 orchestrate the response of pyramidal neurons to neuronal activation. Exposure to novel environment in vivo causes changes in nuclear shape of CA1 hippocampal neurons via a SATB2-dependent mechanism. The activity-driven plasticity of the nuclear envelope requires not only SATB2, but also its protein interactor LEMD2 and the ESCRT-III/VPS4 membrane-remodeling complex. Furthermore, LEMD2 depletion in cortical neurons, similar to SATB2 ablation, affects neuronal activity-dependent regulation of multiple rapid and delayed primary response genes. In human genetic data, LEMD2-regulated genes are enriched for de novo mutations reported in intellectual disability and schizophrenia and are, like SATB2-regulated genes, enriched for common variants associated with schizophrenia and cognitive function. Hence, interactions between SATB2 and the inner nuclear membrane protein LEMD2 influence gene expression programs in pyramidal neurons that are linked to cognitive ability and psychiatric disorder etiology.

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that S. cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast.

The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (Saccharomyces cerevisiae) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis.

ESCRT-III/Vps4 Controls Heterochromatin-Nuclear Envelope Attachments.

Eukaryotic genomes are organized within the nucleus through interactions with inner nuclear membrane (INM) proteins. How chromatin tethering to the INM is controlled in interphase and how this process contributes to subsequent mitotic nuclear envelope (NE) remodeling remains unclear. We have probed these fundamental questions using the fission yeast Schizosaccharomyces japonicus, which breaks and reforms the NE during mitosis. We show that attachments between heterochromatin and the transmembrane Lem2-Nur1 complex at the INM are remodeled in interphase by the ESCRT-III/Vps4 machinery. Failure of ESCRT-III/Vps4 to release Lem2-Nur1 from heterochromatin leads to persistent association of chromosomes with the INM throughout mitosis. At mitotic exit, such trapping of Lem2-Nur1 on heterochromatin prevents it from re-establishing nucleocytoplasmic compartmentalization. Our work identifies the Lem2-Nur1 complex as a substrate for the nuclear ESCRT machinery and explains how the dynamic tethering of chromosomes to the INM is linked to the establishment of nuclear compartmentalization.

TORC1 regulates vacuole membrane composition through ubiquitin- and ESCRT-dependent microautophagy.

Cellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the up-regulation of autophagy and vacuole proteins involved in recycling but also result in the down-regulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Down-regulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.

Plasma membrane tension regulates eisosome structure and function.

Eisosomes are membrane furrows at the cell surface of yeast that have been shown to function in two seemingly distinct pathways, membrane stress response and regulation of nutrient transporters. We found that many stress conditions affect both of these pathways by changing plasma membrane tension and thus the morphology and composition of eisosomes. For example, alkaline stress causes swelling of the cell and an endocytic response, which together increase membrane tension, thereby flattening the eisosomes. The flattened eisosomes affect membrane stress pathways and release nutrient transporters, which aids in their down-regulation. In contrast, glucose starvation or hyperosmotic shock causes cell shrinking, which results in membrane slack and the deepening of eisosomes. Deepened eisosomes are able to trap nutrient transporters and protect them from rapid endocytosis. Therefore, eisosomes seem to coordinate the regulation of both membrane tension and nutrient transporter stability.

The Siderophore Transporter Sit1 Determines Susceptibility to the Antifungal VL-2397.

VL-2397 (previously termed ASP2397) is an antifungal, aluminum-chelating cyclic hexapeptide with a structure analogous to that of ferrichrome-type siderophores, whereby replacement of aluminum by iron was shown to decrease the antifungal activity of this compound. Here, we found that inactivation of an importer for ferrichrome-type siderophores, termed Sit1, renders Aspergillus fumigatus resistant to VL-2397. Moreover, expression of the endogenous sit1 gene under the control of a xylose-inducible promoter (to uncouple sit1 expression from iron repression) combined with C-terminal tagging with a fluorescent protein demonstrated localization of Sit1 in the plasma membrane and xylose-dependent VL-2397 susceptibility. This underlines that Sit1-mediated uptake is essential for VL-2397 susceptibility. Under xylose-induced sit1 expression, VL-2397 also retained antifungal activity after replacing aluminum with iron, which demonstrates that VL-2397 bears antifungal activity independent of cellular aluminum importation. Analysis of sit1 expression indicated that the reduced antifungal activity of the iron-chelated VL-2397 is caused by downregulation of sit1 expression by the imported iron. Furthermore, we demonstrate that defects in iron homeostatic mechanisms modulate the activity of VL-2397. In contrast to A. fumigatus and Candida glabrata, Saccharomyces cerevisiae displays intrinsic resistance to VL-2397 antifungal activity. However, expression of sit1 from A. fumigatus, or its homologue from C. glabrata, resulted in susceptibility to VL-2397, which suggests that the intrinsic resistance of S. cerevisiae is based on lack of uptake and that A. fumigatus, C. glabrata, and S. cerevisiae share an intracellular target for VL-2397.

Endosome and Golgi-associated degradation (EGAD) of membrane proteins regulates sphingolipid metabolism.

Cellular homeostasis requires the ubiquitin-dependent degradation of membrane proteins. This was assumed to be mediated exclusively either by endoplasmic reticulum-associated degradation (ERAD) or by endosomal sorting complexes required for transport (ESCRT)-dependent lysosomal degradation. We identified in Saccharomyces cerevisiae an additional pathway that selectively extracts membrane proteins at Golgi and endosomes for degradation by cytosolic proteasomes. One endogenous substrate of this endosome and Golgi-associated degradation pathway (EGAD) is the ER-resident membrane protein Orm2, a negative regulator of sphingolipid biosynthesis. Orm2 degradation is initiated by phosphorylation, which triggers its ER export. Once on Golgi and endosomes, Orm2 is poly-ubiquitinated by the membrane-embedded "Defective in SREBP cleavage" (Dsc) ubiquitin ligase complex. Cdc48/VCP then extracts ubiquitinated Orm2 from membranes, which is tightly coupled to the proteasomal degradation of Orm2. Thereby, EGAD prevents the accumulation of Orm2 at the ER and in post-ER compartments and promotes the controlled de-repression of sphingolipid biosynthesis. Thus, the selective degradation of membrane proteins by EGAD contributes to proteostasis and lipid homeostasis in eukaryotic cells.

Biogenesis of lysosome-related organelles complex-1 (BORC) regulates late endosomal/lysosomal size through PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate.

Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.

ESCRT and Membrane Protein Ubiquitination.

The ubiquitin-dependent degradation of membrane proteins via the multivesicular body (MVB) pathway requires the Endosomal Sorting Complexes Required for Transport (ESCRT). This molecular machinery is composed of five distinct multi-subunit complexes. On the surface of endosomes, ESCRT-0, -I and -II bind to ubiquitinated membrane proteins, while ESCRT-III and Vps4 bud intraluminal vesicles (ILVs) into the lumen of the endosomes. By working together, ESCRTs package membrane proteins into ILVs and thereby generate MVBs. The fusion of mature MVBs with lysosomes delivers ILVs into the lysosomal lumen where the membrane proteins are degraded. Besides generating ILVs, the ESCRT machinery mediates for topologically related membrane budding processes at the plasma membrane and the nuclear envelop. In this chapter, we briefly discuss membrane protein ubiquitination, endocytosis, and summarize current knowledge on the ESCRT machinery in the MVB pathway.

Functional patchworking at the plasma membrane.

Lipids and proteins are not evenly distributed within the plasma membrane (PM), but instead segregate laterally into many specialized microdomains whose functional relevance is not clear. In this issue, Busto et al (2018) demonstrate that substrate flux through a nutrient transporter drives the lateral relocation of the transporter between specific microdomains at the yeast PM, suggesting that regulating the lateral plasma membrane compartmentalization for individual proteins could be a general process for cellular response to environmental conditions.

The yeast arrestin-related protein Bul1 is a novel actor of glucose-induced endocytosis.

Yeast cells have a remarkable ability to adapt to nutritional changes in their environment. During adaptation, nutrient-signaling pathways drive the selective endocytosis of nutrient transporters present at the cell surface. A current challenge is to understand the mechanistic basis of this regulation. Transporter endocytosis is triggered by their ubiquitylation, which involves the ubiquitin ligase Rsp5 and its adaptors of the arrestin-related family (ART). This step is highly regulated by nutrient availability. For instance, the monocarboxylate transporter Jen1 is ubiquitylated, endocytosed, and degraded upon exposure to glucose. The ART protein Rod1 is required for this overall process; yet Rod1 rather controls Jen1 trafficking later in the endocytic pathway and is almost dispensable for Jen1 internalization. Thus, how glucose triggers Jen1 internalization remains unclear. We report that another ART named Bul1, but not its paralogue Bul2, contributes to Jen1 internalization. Bul1 responds to glucose availability, and preferentially acts at the plasma membrane for Jen1 internalization. Thus, multiple ARTs can act sequentially along the endocytic pathway to control transporter homeostasis. Moreover, Bul1 is in charge of Jen1 endocytosis after cycloheximide treatment, suggesting that the functional redundancy of ARTs may be explained by their ability to interact with multiple cargoes in various conditions.

Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding.

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 s lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

Regulation of Rab5 isoforms by transcriptional and post-transcriptional mechanisms in yeast.

Rab5 GTPases are master regulators of early endosome biogenesis and transport. The genome of Saccharomyces cerevisiae encodes three Rab5 proteins: Vps21, the major isoform, Ypt52 and Ypt53. Here, we show that Vps21 is the most abundant Rab5 protein and Ypt53 is the least abundant. In stressed cells, Ypt53 levels increase but never exceed that of Vps21. Its induction requires the transcription factors Crz1 and Gis1. In growing cells, the expression of Ypt53 is suppressed by post-transcriptional mechanisms mediated by the untranslated regions of the YPT53 mRNA. Based on genetic experiments, these sequences appear to stimulate deadenylation, Pat1-activated decapping and Xrn1-mediated mRNA degradation. Once this regulation is bypassed, Ypt53 protein levels surpass Vps21, and Ypt53 is sufficient to maintain endosomal function and cell growth.

Meeting report - Emerging Concepts in Cell Organization.

New concepts in cell organization emerged in a medieval castle during a snowy week in January 2017 in the middle of the Austrian Alps. The occasion was the 10th Annaberg EMBO workshop in Goldegg am See; organized by Gabriele Seethaler, Catherine Rabouille and Marino Zerial. There were 95 participants, including many who gave talks and presented posters, enjoying a familial and trusting atmosphere that stimulated lively exchange of (unpublished) results, new ideas and thoughts.

ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion.

The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior.