ABSTRACT
Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane.
Subject(s)
Adenosine Triphosphatases/metabolism , Membrane Proteins/metabolism , Mitochondrial Membranes/enzymology , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Conserved Sequence , Hydrolysis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Models, Molecular , Mutation , Protein Domains , Protein Structure, Quaternary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Structure-Activity RelationshipABSTRACT
Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder which lack a stable three-dimensional structure. Interestingly, many of these disordered domains have recently been found to form networks stabilized by weak, multivalent contacts, leading to assembly of protein liquid phases on membrane surfaces. Here we ask how membrane-associated protein liquids impact membrane curvature. We find that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. A simple mechanical model of this process accurately predicts the experimentally measured relationship between the rigidity of the membrane and the diameter of the membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling is not exclusive to structured scaffolds but can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes.
Subject(s)
Cell Membrane/chemistry , Compressive Strength , Membrane Proteins/chemistry , Stress, Mechanical , Humans , Protein ConformationABSTRACT
Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1Δ cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in cisternal maturation kinetics. The arf1Δ mutation causes early Golgi cisternae to mature more slowly and less frequently, but does not alter the maturation of late Golgi cisternae. These changes quantitatively explain why late Golgi cisternae are fewer in number and correspondingly larger. With a stacked Golgi, similar changes in maturation kinetics could be used by the cell to modulate the number of cisternae per stack. Thus, the rates of processes that transform a maturing compartment can determine compartmental size and copy number.
Subject(s)
ADP-Ribosylation Factor 1/genetics , Gene Expression Regulation, Fungal , Golgi Apparatus/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , ADP-Ribosylation Factor 1/deficiency , Biological Transport , Golgi Apparatus/ultrastructure , Mutation , Myristic Acids/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The Golgi apparatus contains multiple classes of cisternae that differ in structure, composition, and function, but there is no consensus about the number and definition of these classes. A useful way to classify Golgi cisternae is according to the trafficking pathways by which the cisternae import and export components. By this criterion, we propose that Golgi cisternae can be divided into three classes that correspond to functional stages of maturation. First, cisternae at the cisternal assembly stage receive COPII vesicles from the ER and recycle components to the ER in COPI vesicles. At this stage, new cisternae are generated. Second, cisternae at the carbohydrate synthesis stage exchange material with one another via COPI vesicles. At this stage, most of the glycosylation and polysaccharide synthesis reactions occur. Third, cisternae at the carrier formation stage produce clathrin-coated vesicles and exchange material with endosomes. At this stage, biosynthetic cargo proteins are packaged into various transport carriers, and the cisternae ultimately disassemble. Discrete transitions occur as a cisterna matures from one stage to the next. Within each stage, the structure and composition of a cisterna can evolve, but the trafficking pathways remain unchanged. This model offers a unified framework for understanding the properties of the Golgi in diverse organisms.
Subject(s)
Golgi Apparatus/chemistry , Golgi Apparatus/physiology , Models, Biological , Animals , Biological Transport , Endoplasmic Reticulum/metabolism , Glycosylation , Polysaccharides/biosynthesis , Polysaccharides/chemistryABSTRACT
Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple labs have reported that early endocytic proteins form liquid-like condensates, which provide a flexible platform for the efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic sites. In live cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitinase enzyme destabilized nascent endocytic sites within minutes. These results suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic recycling.
ABSTRACT
Endocytosis is a fine-tuned mechanism of cellular communication through which cells internalize molecules on the plasma membrane, such as receptors and their bound ligands. Through receptor clustering in endocytic pits, recruitment of active receptors to different endocytic routes and their trafficking towards different fates, endocytosis modulates cell signaling and ultimately leads to a variety of biological responses. Many studies have focused their attention on specialized endocytic mechanisms depending on the nature of the internalizing cargo and cellular context, distinct sets of coat proteins, endocytic adaptors and membrane lipids. Here, we review recent advances in our understanding of the principles underlying endocytic vesicle formation, integrating both biochemical and biophysical factors, with a particular focus on intrinsically disordered regions (IDRs) creating weakly interconnected protein networks assembled through liquid-liquid phase separation (LLPS) and driving membrane bending especially in clathrin-mediated endocytosis (CME). We finally discuss how these properties impinge on receptor fate and signaling.
Subject(s)
Clathrin , Endocytosis , Biophysics , Cell Membrane/metabolism , Clathrin/metabolism , Transport Vesicles/metabolismABSTRACT
The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Endocytosis , Golgi Apparatus/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Cell Membrane/metabolism , Kinetics , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , trans-Golgi Network/metabolismABSTRACT
During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As initiators of endocytosis, Eps15 and Fcho1/2 concentrate downstream components, while permitting dynamic rearrangement within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1/2 rely on weak, liquid-like interactions to catalyse endocytosis. In vitro, these weak interactions promote the assembly of protein droplets with liquid-like properties. To probe the physiological role of these liquid-like networks, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15-knockout cells. By contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels, which stalled vesicle budding, probably owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of initiator proteins provides an optimal catalytic platform for endocytosis.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cell Membrane/genetics , Fatty Acid-Binding Proteins/genetics , Membrane Proteins/genetics , Transport Vesicles/genetics , Animals , Calcium-Binding Proteins/genetics , Catalysis , Clathrin/genetics , Endocytosis/genetics , Humans , Mice , Phosphoproteins/geneticsABSTRACT
Intracellular trafficking requires extensive changes in membrane morphology. Cells use several distinct molecular factors and physical cues to remodel membranes. Here, we highlight recent advances in identifying the biophysical mechanisms of membrane curvature generation. In particular, we focus on the cooperation of molecular and physical drivers of membrane bending during three stages of vesiculation: budding, cargo selection, and scission. Taken together, the studies reviewed here emphasize that, rather than a single dominant mechanism, several mechanisms typically work in parallel during each step of membrane remodeling. Important challenges for the future of this field are to understand how multiple mechanisms work together synergistically and how a series of stochastic events can be combined to achieve a deterministic result-assembly of the trafficking vesicle.
Subject(s)
Biophysical Phenomena , Cell Membrane/metabolism , Actins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Macromolecular Substances/metabolism , Protein TransportABSTRACT
Many critical biological events, including biochemical signaling, membrane traffic, and cell motility, originate at membrane surfaces. Each such event requires that members of a specific group of proteins and lipids rapidly assemble together at a specific site on the membrane surface. Understanding the biophysical mechanisms that stabilize these assemblies is critical to decoding and controlling cellular functions. In this article, we review progress toward a quantitative biophysical understanding of the mechanisms that drive membrane heterogeneity and organization. We begin from a physical perspective, reviewing the fundamental principles and key experimental evidence behind each proposed mechanism. We then shift to a biological perspective, presenting key examples of the role of heterogeneity in biology and asking which physical mechanisms may be responsible. We close with an applied perspective, noting that membrane heterogeneity provides a novel therapeutic target that is being exploited by a growing number of studies at the interface of biology, physics, and engineering.
Subject(s)
Cell Membrane/metabolism , Biophysical Phenomena , Humans , Signal TransductionABSTRACT
The endomembrane system consists of the secretory and endocytic pathways, which communicate by transport to and from the trans-Golgi network (TGN). In mammalian cells, the endocytic pathway includes early, late, and recycling endosomes. In budding yeast, different types of endosomes have been described, but the organization of the endocytic pathway has remained unclear. We performed a spatial and temporal analysis of yeast endosomal markers and endocytic cargoes. Our results indicate that the yeast TGN also serves as an early and recycling endosome. In addition, as previously described, yeast contains a late or prevacuolar endosome (PVE). Endocytic cargoes localize to the TGN shortly after internalization, and manipulations that perturb export from the TGN can slow the passage of endocytic cargoes to the PVE. Yeast apparently lacks a distinct early endosome. Thus, yeast has a simple endocytic pathway that may reflect the ancestral organization of the endomembrane system.
Subject(s)
Cell Membrane/metabolism , Endocytosis/physiology , Endosomes/metabolism , Golgi Apparatus/metabolism , Saccharomycetales/metabolism , trans-Golgi Network/metabolism , Biological Transport , Saccharomycetales/growth & developmentABSTRACT
Deconvolution is typically used to sharpen fluorescence images, but when the signal-to-noise ratio is low, the primary benefit is reduced noise and a smoother appearance of the fluorescent structures. 3D time-lapse (4D) confocal image sets can be improved by deconvolution. However, when the confocal signals are very weak, the popular Huygens deconvolution software erases fluorescent structures that are clearly visible in the raw data. We find that this problem can be avoided by prefiltering the optical sections with a Gaussian blur. Analysis of real and simulated data indicates that the Gaussian blur prefilter preserves meaningful signals while enabling removal of background noise. This approach is very simple, and it allows Huygens to be used with 4D imaging conditions that minimize photodamage.
ABSTRACT
Yeast cells are well suited to visualizing organelles by 4D confocal microscopy. Typically, one or more cellular compartments are labeled with a fluorescent protein or dye, and a stack of confocal sections spanning the entire cell volume is captured every few seconds. Under appropriate conditions, organelle dynamics can be observed for many minutes with only limited photobleaching. Images are captured at a relatively low signal-to-noise ratio and are subsequently processed to generate movies that can be analyzed and quantified. Here, we describe methods for acquiring and processing 4D data using conventional scanning confocal microscopy.
Subject(s)
Image Processing, Computer-Assisted/methods , Organelles , Saccharomyces cerevisiae/cytology , Microscopy, Confocal/methodsABSTRACT
FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP.
ABSTRACT
Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76kbp between two previous gaps on chromosome 1 and another 134kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5kbp found in some strains of P. pastoris.
Subject(s)
DNA, Fungal/genetics , Genome, Fungal/genetics , Pichia/genetics , Alternative Splicing , Centromere/genetics , Genetic Engineering , Plasmids/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Transcriptome/geneticsABSTRACT
COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.