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The sequential action of five distinct e ndosomal‐ s orting c omplex r equired for t ransport (ESCRT) complexes is required for the lysosomal downregulation of cell surface receptors through the m ulti v esicular b ody (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.

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.

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for e ndosome and G olgi a ssociated proteasomal d egradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition. At the Golgi, the Dsc ubiquitin ligase complex targets proteins with shorter transmembrane domains for proteasomal or lysosomal degradation. This quality control at the sorting center of cells restricts the uncontrolled spreading of orphaned proteins.

The degradation and recycling of cellular components is essential for cell growth and survival. Here we show how selective and non-selective lysosomal protein degradation pathways cooperate to ensure cell survival upon nutrient limitation. A quantitative analysis of starvation-induced proteome remodeling in yeast reveals comprehensive changes already in the first three hours. In this period, many different integral plasma membrane proteins undergo endocytosis and degradation in vacuoles via the multivesicular body (MVB) pathway. Their degradation becomes essential to maintain critical amino acids levels that uphold protein synthesis early during starvation. This promotes cellular adaptation, including the de novo synthesis of vacuolar hydrolases to boost the vacuolar catabolic activity. This order of events primes vacuoles for the efficient degradation of bulk cytoplasm via autophagy. Hence, a catabolic cascade including the coordinated action of the MVB pathway and autophagy is essential to enter quiescence to survive extended periods of nutrient limitation. Yeast and other organisms have evolved to survive extended periods of starvation by digesting their own proteins and other cell materials and thereby recycle them into new proteins and structures. One way in which these cell materials can be destroyed is by a process called autophagy. A membrane forms around the cell material to isolate it from the rest of the cell. In yeast, the resulting structure fuses with a cell compartment called the vacuole, which contains enzymes that break down the cargo into smaller molecules that can be re-used by the cell. When cells experience starvation, autophagy is not very selective in what it destroys and so it is tightly controlled to avoid damaging important structures in healthy cells. Alongside autophagy, specific proteins in the membrane surrounding a yeast cell can be targeted for destruction by another process called the MVB pathway. Certain membrane proteins are tagged with a small protein called ubiquitin, which leads them to being selectively incorporated into cell compartments called MVBs that then go on to fuse with the vacuole. However, it is not clear how the MVB pathway and autophagy may cooperate to enable the cell to survive periods of starvation. Here, Müller et al. monitored the changes in the proteins present in yeast cells during a period of starvation. The experiments show that many different membrane proteins in the yeast cells were destroyed via the MVB pathway within three hours of the removal of their food source. This was essential to allow the cells to carry on producing new proteins at this early stage in starvation. These new proteins included the enzymes found in vacuoles, which increased the ability of the cells to break down the proteins and other cell materials that were transported there via autophagy. These findings show how the MVB pathway and autophagy are co-ordinated to allow cells to survive periods of starvation. The next challenge is to work out how the MVB pathway is regulated at the molecular level in response to fluctuations in nutrient availability.

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.

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. Cells produce thousands of different proteins with a variety of different roles in the body. Some proteins, for example the hormone insulin, perform roles outside of the cell and are released from cells in a process that has several stages. In the first step, newly-made insulin and many other “secretory” proteins enter a compartment called the endoplasmic reticulum. Once inside, these proteins can then be loaded into other compartments and transported to the edge of the cell. There is another class of secretory proteins that are released from the cell without first entering the endoplasmic reticulum, in a process termed “unconventional protein secretion”. A protein called Acb1 is released from yeast cells in this manner. Previous research identified a compartment that might be involved in this process. However, it is not clear how this compartment (named CUPS) forms, and what role it plays in unconventional protein secretion. Curwin et al. investigated how CUPS form in yeast cells, and whether the compartment contains Acb1 proteins. The experiments reveal that after CUPS form they need to mature into a form that is involved in the release of Acb1 proteins from the cell. This maturation process involves some, but not all, of the same genes as those involved in producing another type of compartment in cells called a multivesicular body. Acb1 is only found in the mature CUPS and multivesicular bodies are not involved in the release of this protein from the cell. Curwin et al.’s findings shed some light on how Acb1 and other secretory proteins can be released from cells without involving the endoplasmic reticulum. Future challenges are to reveal how CUPS capture cargo and find out how Acb1 leaves the CUPS to exit the cell.

Signaling pathways in eukaryotic cells are often controlled by the formation of specific signaling complexes, which are coordinated by scaffold and adaptor proteins. Elucidating their molecular architecture is essential to understand the spatial and temporal regulation of cellular signaling. p14 and MP1 form a tight (Kd=12.8 nM) endosomal adaptor/scaffold complex, which regulates mitogen-activated protein kinase (MAPK) signaling. Here, we present the 1.9-Å crystal structure of a biologically functional p14/MP1 complex. The overall topology of the individual MP1 and p14 proteins is almost identical, having a central five-stranded β-sheet sandwiched between a two-helix and a one-helix layer. Formation of the p14/MP1 heterodimer proceeds by β-sheet augmentation and yields a unique, almost symmetrical, complex with several potential protein-binding sites on its surface. Mutational analysis allowed identification of the p14 endosomal adaptor motif, which seems to orient the complex relative to the endosomal membrane. Two highly conserved and hydrophobic protein-binding sites are located on the opposite \"cytoplasmic\" face of the p14/MP1 heterodimer and might therefore function as docking sites for the target proteins extracellular regulated kinase (ERK) 1 and MAPK/ERK kinase 1. Furthermore, detailed sequence analyses revealed that MP1/p14, together with profilins, define a protein superfamily of small subcellular adaptor proteins, named ProflAP. Taken together, the presented work provides insight into the spatial regulation of MAPK signaling, illustrating how p14 and MP1 collaborate as an endosomal adaptor/scaffold complex.

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 [alpha]-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 [alpha]-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

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.

In eukaryotes chromosomes are compartmentalized within the nucleus delimited by the double membrane of the nuclear envelope (NE). Defects in the function and structure of the NE are linked to disease. During interphase, the NE organizes the genome and regulates its expression. As cells enter mitosis, chromosomes are released from the NE, which is then remodelled to form the daughter nuclei at mitotic exit. Interactions between the NE and chromatin underpinning both interphase and post-mitotic NE functions are executed by inner nuclear membrane (INM) proteins such as members of the evolutionarily conserved chromatin-binding LEM-domain family. How chromatin tethering by these transmembrane proteins is controlled in interphase and if such a regulation contributes to subsequent NE dynamics in mitosis remains unclear. Here we probe these fundamental questions using an emerging model organism, 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 are continuously remodelled in interphase by the ESCRT-III/AAA-ATPase Vps4 machinery. ESCRT-III/Vps4 mediates the release of Lem2-Nur1 from heterochromatin as a prerequisite for the timely progression through mitosis. Failure in this process leads to persistent association of chromosomes with the INM, which prevents Lem2-Nur1 from re-localizing to the sites of NE sealing around the mitotic spindle and severely delays re-establishment of nucleocytoplasmic compartmentalization. Our work establishes the INM transmembrane Lem2-Nur1 complex as a substrate for ESCRT-III/Vps4 to couple dynamic tethering of chromosomes to the INM with the establishment of nuclear compartmentalization.