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.

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.

Functional reconstitution of ESCRT-III assembly and disassembly.

Receptor downregulation in the MVB pathway is mediated by the ESCRT complexes. ESCRT-III is composed of four protein subunits that are monomeric in the cytosol and oligomerize into a protein lattice only upon membrane binding. Recent studies have shown that the ESCRT-III protein Snf7 can form a filament by undergoing homo-oligomerization. To examine the role of membrane binding and of interactions with other ESCRT components in initiating Snf7 oligomerization, we used fluorescence spectroscopy to directly detect and characterize the assembly of the Snf7 oligomer on liposomes using purified ESCRT components. The observed fluorescence changes reveal an obligatory sequence of membrane-protein and protein-protein interactions that generate the active conformation of Snf7. Also, we demonstrate that ESCRT-III assembly drives membrane deformation. Furthermore, using an in vitro disassembly assay, we directly demonstrate that Vps24 and Vps2 function as adaptors in the ATP-dependent membrane disassembly of the ESCRT-III complex by recruiting the AAA ATPase Vps4.

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.

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 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.

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.

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.

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.

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 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.

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System.

The late endosomal adaptor protein LAMTOR2/p14 is essential for tissue homeostasis by controlling MAPK and mTOR signaling, which in turn regulate cell growth and proliferation, migration and spreading. Moreover, LAMTOR2 critically controls architecture and function of the endocytic system, including epidermal growth factor receptor (EGFR) degradation in lysosomes, positioning of late endosomes and defense against intracellular pathogens. Here we describe the multifaceted ultrastructural phenotype of the endo/lysosomal system of LAMTOR2-deficient mouse embryonic fibroblasts. Quantitative (immuno-)electron microscopy of cryo-fixed samples revealed significantly reduced numbers of recycling tubules emanating from maturing multivesicular bodies (MVB). Instead, a distinct halo of vesicles surrounded MVB, tentatively interpreted as detached, jammed recycling tubules. These morphological changes in LAMTOR2-deficient cells correlated with the presence of growth factors (e.g. EGF), but were similarly induced in control cells by inactivating mTOR. Furthermore, proper transferrin receptor trafficking and recycling were apparently dependent on an intact LAMTOR complex. Finally, a severe imbalance in the relative proportions of endo/lysosomes was found in LAMTOR2-deficient cells, resulting from increased amounts of mature MVB and (autophago)lysosomes. These observations suggest that the LAMTOR/Ragulator complex is required not only for maintaining the homeostasis of endo/lysosomal subpopulations but also contributes to the proper formation of MVB-recycling tubules, and regulation of membrane/cargo recycling from MVB.

Quantitative proteomics using ultralow flow capillary electrophoresis-mass spectrometry.

In this work, we evaluate the incorporation of an ultralow flow interface for coupling capillary electrophoresis (CE) and mass spectrometry (MS), in combination with reversed-phase high-pressure liquid chromatography (HPLC) fractionation as an alternate workflow for quantitative proteomics. Proteins, extracted from a SILAC (stable isotope labeling by amino acids in cell culture) labeled and an unlabeled yeast strain were mixed and digested enzymatically in solution. The resulting peptides were fractionated using RP-HPLC and analyzed by CE-MS yielding a total of 28 538 quantified peptides that correspond to 3 272 quantified proteins. CE-MS analysis was performed using a neutral capillary coating, providing the highest separation efficiency at ultralow flow conditions (<10 nL/min). Moreover, we were able to demonstrate that CE-MS is a powerful method for the identification of low-abundance modified peptides within the same sample. Without any further enrichment strategies, we succeeded in quantifying 1 371 phosphopeptides present in the CE-MS data set and found 49 phosphopeptides to be differentially regulated in the two yeast strains. Including acetylation, phosphorylation, deamidation, and oxidized forms, a total of 8 106 modified peptides could be identified in addition to 33 854 unique peptide sequences found. The work presented here shows the first quantitative proteomics approach that combines SILAC labeling with CE-MS analysis.

The role of the endosomal sorting complexes required for transport (ESCRT) in tumorigenesis.

The endosomal sorting complexes required for transport (ESCRT) are needed for three distinct cellular functions in higher eukaryotes: (i) Multivesicular body formation for the degradation of transmembrane proteins in lysosomes, (ii) midbody abscission during cytokinesis and (iii) retroviral budding. Not surprisingly, loss of ESCRT function has severe consequences, which include the failure to down-regulate growth factor receptors leading to deregulated mitogenic signaling. While it is clear that the function of the ESCRT machinery is important for embryonic development, its role in cancer is more controversial. Various experimental approaches in different model organisms arrive at partially divergent conclusions regarding the contribution of ESCRTs to tumorigenesis. Therefore the aim of this review is to provide an overview on different model systems used to study the role of the ESCRT machinery in cancer development, to highlight common grounds and present certain controversies in the field.

ESCRT-II coordinates the assembly of ESCRT-III filaments for cargo sorting and multivesicular body vesicle formation.

The sequential action of five distinct endosomal-sorting complex required for transport (ESCRT) complexes is required for the lysosomal downregulation of cell surface receptors through the multivesicular body (MVB) pathway. On endosomes, the assembly of ESCRT-III is a highly ordered process. We show that the length of ESCRT-III (Snf7) oligomers controls the size of MVB vesicles and addresses how ESCRT-II regulates ESCRT-III assembly. The first step of ESCRT-III assembly is mediated by Vps20, which nucleates Snf7/Vps32 oligomerization, and serves as the link to ESCRT-II. The ESCRT-II subunit Vps25 induces an essential conformational switch that converts inactive monomeric Vps20 into the active nucleator for Snf7 oligomerization. Each ESCRT-II complex contains two Vps25 molecules (arms) that generate a characteristic Y-shaped structure. Mutant 'one-armed' ESCRT-II complexes with a single Vps25 arm are sufficient to nucleate Snf7 oligomerization. However, these oligomers cannot execute ESCRT-III function. Both Vps25 arms provide essential geometry for the assembly of a functional ESCRT-III complex. We propose that ESCRT-II serves as a scaffold that nucleates the assembly of two Snf7 oligomers, which together are required for cargo sequestration and vesicle formation during MVB sorting.

A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14.

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3' untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

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.

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.

p14-MP1-MEK1 signaling regulates endosomal traffic and cellular proliferation during tissue homeostasis.

The extracellular signal-regulated kinase (ERK) cascade regulates proliferation, differentiation, and survival in multicellular organisms. Scaffold proteins regulate intracellular signaling by providing critical spatial and temporal specificity. The scaffold protein MEK1 (mitogen-activated protein kinase and ERK kinase 1) partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in mice, we now demonstrate that the p14-MP1-MEK1 signaling complex regulates late endosomal traffic and cellular proliferation. This function its essential for early embryogenesis and during tissue homeostasis, as revealed by epidermis-specific deletion of p14. These findings show that endosomal p14-MP1-MEK1 signaling has a specific and essential function in vivo and, therefore, indicate that regulation of late endosomal traffic by extracellular signals is required to maintain tissue homeostasis.