AP‐1A Controls Secretory Granule Biogenesis and Trafficking of Membrane Secretory Granule Proteins

The adaptor protein 1A complex (AP‐1A) transports cargo between the trans‐Golgi network (TGN) and endosomes. In professional secretory cells, AP‐1A also retrieves material from immature secretory granules (SGs). The role of AP‐1A in SG biogenesis was explored using AtT‐20 corticotrope tumor cells expressing reduced levels of the AP‐1A μ1A subunit. A twofold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non‐condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue‐stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α‐amidating monooxygenase‐1 (PAM‐1), integral membrane enzymes that enter immature SGs. The non‐condensing SGs contained POMC products and PAM‐1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM‐1 into PHM was unaltered, but PHM basal secretion was increased in sh‐μ1A PAM‐1 cells. Despite lacking a canonical AP‐1A binding motif, yeast two‐hybrid studies demonstrated an interaction between the PAM‐1 cytosolic domain and AP‐1A. Coimmunoprecipitation experiments with PAM‐1 mutants revealed an influence of the luminal domains of PAM‐1 on this interaction. Thus, AP‐1A is crucial for normal SG biogenesis, function and composition.      

Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation

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LYST Controls the Biogenesis of the Endosomal Compartment Required for Secretory Lysosome Function

Chediak–Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome‐like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling‐late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal‐sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13‐4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin‐containing vesicles into secretory cytotoxic granules.      

The filament-forming protein Pil1 assembles linear eisosomes in fission yeast

The cortical cytoskeleton mediates a range of cellular activities such as endocytosis, cell motility, and the maintenance of cell rigidity. Traditional polymers, including actin, microtubules, and septins, contribute to the cortical cytoskeleton, but additional filament systems may also exist. In yeast cells, cortical structures called eisosomes generate specialized domains termed MCCs to cluster specific proteins at sites of membrane invaginations. Here we show that the core eisosome protein Pil1 forms linear cortical filaments in fission yeast cells and that purified Pil1 assembles into filaments in vitro. In cells, Pil1 cortical filaments are excluded from regions of cell growth and are independent of the actin and microtubule cytoskeletons. Pil1 filaments assemble slowly at the cell cortex and appear stable by time-lapse microscopy and fluorescence recovery after photobleaching. This stability does not require the cell wall, but Pil1 and the transmembrane protein Fhn1 colocalize and are interdependent for localization to cortical filaments. Increased Pil1 expression leads to cytoplasmic Pil1 rods that are stable and span the length of cylindrical fission yeast cells. We propose that Pil1 is a novel component of the yeast cytoskeleton, with implications for the role of filament assembly in the spatial organization of cells.     

酵母细胞中Pil1的磷酸化与细胞压力抵抗的关系

在外界因素处理下,细胞将启动一系列保护措施以适应各种环境改变,磷酸化调节是蛋白功能调节的主要方式. 为了探讨酵母细胞中Pil1的磷酸化与细胞压力抵抗的关系,实验应用Pil1突变细胞检测在过氧化氢或热处理后细胞的生长情况,用免疫印记法检测热处理后Pil1的表达. 结果表明,相比野生细胞,Pil1突变细胞对抗过氧化氢和热的能力强,热处理后 Pil1的磷酸化水平增高, Pil1的丝氨酸273对于其磷酸化发生至关重要.     

Eisosome proteins assemble into a membrane scaffold

Spatial organization of membranes into domains of distinct protein and lipid composition is a fundamental feature of biological systems. The plasma membrane is organized in such domains to efficiently orchestrate the many reactions occurring there simultaneously. Despite the almost universal presence of membrane domains, mechanisms of their formation are often unclear. Yeast cells feature prominent plasma membrane domain organization, which is at least partially mediated by eisosomes. Eisosomes are large protein complexes that are primarily composed of many subunits of two Bin-Amphiphysin-Rvs domain-containing proteins, Pil1 and Lsp1. In this paper, we show that these proteins self-assemble into higher-order structures and bind preferentially to phosphoinositide-containing membranes. Using a combination of electron microscopy approaches, we generate structural models of Pil1 and Lsp1 assemblies, which resemble eisosomes in cells. Our data suggest that the mechanism of membrane organization by eisosomes is mediated by self-assembly of its core components into a membrane-bound protein scaffold with lipid-binding specificity.     

Disulfide Bond Formation: Sulfhydryl Oxidase ALR Controls Mitochondrial Biogenesis of Human MIA40

The conserved MIA pathway is responsible for the import and oxidative folding of proteins destined for the intermembrane space of mitochondria. In contrast to a wealth of information obtained from studies with yeast, the function of the MIA pathway in higher eukaryotes has remained enigmatic. Here, we took advantage of the molecular understanding of the MIA pathway in yeast and designed a model of the human MIA pathway. The yeast model for MIA consists of two critical components, the disulfide bond carrier Mia40 and sulfhydryl oxidase Erv1/ALR. Human MIA40 and ALR substituted for their yeast counterparts in the essential function for the oxidative biogenesis of mitochondrial intermembrane space proteins. In addition, the sulfhydryl oxidases ALR/Erv1 were found to be involved in the mitochondrial localization of human MIA40. Furthermore, the defective accumulation of human MIA40 in mitochondria underlies a recently identified disease that is caused by amino acid exchange in ALR. Thus, human ALR is an important factor that controls not only the ability of MIA40 to bind and oxidize protein clients but also the localization of human MIA40 in mitochondria.     

Eisosomes Are Dynamic Plasma Membrane Domains Showing Pil1-Lsp1 Heteroligomer Binding Equilibrium

Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits.     

ArfGAP1 Activity and COPI Vesicle Biogenesis

Golgi-derived coat protein I (COPI) vesicles mediate transport in the early secretory pathway. The minimal machinery required for COPI vesicle formation from Golgi membranes in vitro consists of (i) the hetero-heptameric protein complex coatomer, (ii) the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) and (iii) transmembrane proteins that function as coat receptors, such as p24 proteins. Various and opposing reports exist on a role of ArfGAP1 in COPI vesicle biogenesis. In this study, we show that, in contrast to data in the literature, ArfGAP1 is not required for COPI vesicle formation. To investigate roles of ArfGAP1 in vesicle formation, we titrated the enzyme into a defined reconstitution assay to form and purify COPI vesicles. We find that catalytic amounts of Arf1GAP1 significantly reduce the yield of purified COPI vesicles and that Arf1 rather than ArfGAP1 constitutes a stoichiometric component of the COPI coat. Combining the controversial reports with the results presented in this study, we suggest a novel role for ArfGAP1 in membrane trafficking.     

Biogenesis of the yeast cytochrome bc1 complex

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.     

Biogenesis and adhesion of type 1 and P pili

BackgroundUropathogenic Escherichia coli (UPEC) cause urinary tract infections (UTIs) in approximately 50% of women. These bacteria use type 1 and P pili for host recognition and attachment. These pili are assembled by the chaperone-usher pathway of pilus biogenesis.Scope of reviewThe review examines the biogenesis and adhesion of the UPEC type 1 and P pili. Particular emphasis is drawn to the role of the outer membrane usher protein. The structural properties of the complete pilus are also examined to highlight the strength and functionality of the final assembly.Major conclusionsThe usher orchestrates the sequential addition of pilus subunits in a defined order. This process follows a subunit-incorporation cycle which consists of four steps: recruitment at the usher N-terminal domain, donor-strand exchange with the previously assembled subunit, transfer to the usher C-terminal domains and translocation of the nascent pilus.Adhesion by the type 1 and P pili is strengthened by the quaternary structure of their rod sections. The rod is endowed with spring-like properties which provide mechanical resistance against urine flow. The distal adhesins operate differently from one another, targeting receptors in a specific manner.The biogenesis and adhesion of type 1 and P pili are being therapeutically targeted, and efforts to prevent pilus growth or adherence are described.General significanceThe combination of structural and biochemical study has led to the detailed mechanistic understanding of this membrane spanning nano-machine. This can now be exploited to design novel drugs able to inhibit virulence. This is vital in the present era of resurgent antibiotic resistance. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.     

The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis

The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1-deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence microscopy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1-deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa procathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in phagolysosome biogenesis.     

Eisosome Ultrastructure and Evolution in Fungi,Microalgae, and Lichens

Eisosomes are among the few remaining eukaryotic cellular differentations that lack a defined function(s). These trough-shaped invaginations of the plasma membrane have largely been studied in Saccharomyces cerevisiae, in which their associated proteins, including two BAR domain proteins, have been identified, and homologues have been found throughout the fungal radiation. Using quick-freeze deep-etch electron microscopy to generate high-resolution replicas of membrane fracture faces without the use of chemical fixation, we report that eisosomes are also present in a subset of red and green microalgae as well as in the cysts of the ciliate Euplotes. Eisosome assembly is closely correlated with both the presence and the nature of cell walls. Microalgal eisosomes vary extensively in topology and internal organization. Unlike fungi, their convex fracture faces can carry lineage-specific arrays of intramembranous particles, and their concave fracture faces usually display fine striations, also seen in fungi, that are pitched at lineage-specific angles and, in some cases, adopt a broad-banded patterning. The conserved genes that encode fungal eisosome-associated proteins are not found in sequenced algal genomes, but we identified genes encoding two algal lineage-specific families of predicted BAR domain proteins, called Green-BAR and Red-BAR, that are candidate eisosome organizers. We propose a model for eisosome formation wherein (i) positively charged recognition patches first establish contact with target membrane regions and (ii) a (partial) unwinding of the coiled-coil conformation of the BAR domains then allows interactions between the hydrophobic faces of their amphipathic helices and the lipid phase of the inner membrane leaflet, generating the striated patterns.     

Biogenesis of the T1-S1 linker of voltage-gated K+ channels

In the model derived from the crystal structure of Kv1.2, a six-transmembrane voltage-gated potassium channel, the linker between a cytosolic tetramerization domain, T1, and the first transmembrane segment, S1, is projected radially outward from the channel's central axis. This T1-S1 linker was modeled as two polyglycine helices to accommodate the residues between T1 and S1 [Long et al. (2005) Science 309, 897-903]; however, the structure of this linker is not known. Here, we investigate whether a compact secondary structure of the T1-S1 linker exists at an early stage of Kv channel biogenesis. We have used a mass-tagging accessibility assay to report the biogenesis of secondary structure for three consecutive regions of Kv1.3, a highly homologous isoform of Kv1.2. The three regions include the T1-S1 linker and its two flanking regions, alpha5 of the T1 domain and S1. Both alpha5 and S1 manifest compact structures (helical) inside the ribosomal exit tunnel, whereas the T1-S1 linker does not. Moreover, the location of the peptide in the tunnel influences compaction.     

Biogenesis of monoterpenes

Cell suspension cultures of Muscat de Frontignan grapes Vitis vinifera L. are able to convert citral (a mixture of neral and geranial) into the corresponding monoterpenic alcohols, nerol and geraniol. The geraniol formed is esterified into geranyl acetate. Bioconversion of nerol or geraniol added alone to the cell suspension was also studied. Interconversions between these different monoterpenic compounds are described and discussed.