scholarly journals New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells

2019 ◽  
Vol 218 (11) ◽  
pp. 3861-3879 ◽  
Author(s):  
Laia Bassaganyas ◽  
Stephanie J. Popa ◽  
Max Horlbeck ◽  
Claudia Puri ◽  
Sarah E. Stewart ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Basic Principles ◽  
Golgi Membranes ◽  
Genome Wide ◽  
A Genome ◽  
Pathway Function ◽  
Powerful Strategy

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.

scholarly journals New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells

2019 ◽  
Author(s):  
Laia Bassaganyas ◽  
Stephanie J. Popa ◽  
Max Horlbeck ◽  
Anupama Ashok ◽  
Sarah E. Stewart ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Basic Principles ◽  
Golgi Membranes ◽  
Genome Wide ◽  
A Genome ◽  
Powerful Strategy ◽  
Shed Light

AbstractProtein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathways function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to the Golgi complex. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion, and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.

scholarly journals Conserved function of ether lipids and sphingolipids in the early secretory pathway

2019 ◽  
Author(s):  
Noemi Jiménez-Rojo ◽  
Manuel D. Leonetti ◽  
Valeria Zoni ◽  
Adai Colom ◽  
Suihan Feng ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Cell Biology ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Lipid Synthesis ◽  
Chemical Properties ◽  
Ether Lipid ◽  
Ether Lipids ◽  
Early Secretory Pathway ◽  
A Genome

ABSTRACTSphingolipids have been shown to play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, where depletion of one of these lipid types resulted in increases in the other, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as GPI anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.

scholarly journals Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein β-Isoform to the MammalianTrans-Golgi Network

2006 ◽  
Vol 17 (6) ◽  
pp. 2498-2512 ◽  
Author(s):  
Scott E. Phillips ◽  
Kristina E. Ile ◽  
Malika Boukhelifa ◽  
Richard P.H. Huijbregts ◽  
Vytas A. Bankaitis
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Membrane Binding ◽  
Bound State ◽  
Missense Mutations ◽  
Cis Acting ◽  
Domain Mapping ◽  
Phosphatidylinositol Transfer ◽  
Golgi Network

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITPβ localization in mammalian cells. We demonstrate PITPβ localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITPβ. Domain mapping analyses show the targeting information within PITPβ consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W202W203motif). Combination of the specificity elements with the W202W203motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITPβ association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITPβ isoform with a naturally occurring S262Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITPβ localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms δ or ε. These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser262is required for PITPβ targeting to Golgi membranes.

Controlling quality and amount of mitochondria by mitophagy: insights into the role of ubiquitination and deubiquitination

2016 ◽  
Vol 397 (7) ◽  
pp. 637-647 ◽  
Author(s):  
Tao Tan ◽  
Marcel Zimmermann ◽  
Andreas S. Reichert
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Fission ◽  
Baker’S Yeast ◽  
Negative Regulator ◽  
Mitochondrial Outer Membrane ◽  
Baker's Yeast ◽  
Genome Wide ◽  
A Genome ◽  
Autophagy Pathway ◽  
Quantity Control

Abstract Mitophagy is a selective autophagy pathway conserved in eukaryotes and plays an essential role in mitochondrial quality and quantity control. Mitochondrial fission and fusion cycles maintain a certain amount of healthy mitochondria and allow the isolation of damaged mitochondria for their elimination by mitophagy. Mitophagy can be classified into receptor-dependent and ubiquitin-dependent pathways. The mitochondrial outer membrane protein Atg32 is identified as the only known receptor for mitophagy in baker’s yeast, whereas mitochondrial proteins FUNDC1, NIX/BNIP3L, BNIP3 and Bcl2L13 are recognized as mitophagy receptors in mammalian cells. Earlier studies showed that ubiquitination and deubiquitination occurs in yeast, yet there is no direct evidence for an ubiquitin-dependent mitophagy pathway in this organism. In contrast, a ubiquitin-/PINK1-/Parkin-dependent mitophagy pathway was unraveled and was extensively characterized in mammals in recent years. Recently, a quantitative method termed synthetic quantitative array (SQA) technology was developed to identify modulators of mitophagy in baker’s yeast on a genome-wide level. The Ubp3-Bre5 deubiquitination complex was found as a negative regulator of mitophagy while promoting other autophagic pathways. Here we discuss how ubiquitination and deubiquitination regulates mitophagy and other selective forms of autophagy and what argues for using baker’s yeast as a model to study the ubiquitin-dependent mitophagy pathway.

scholarly journals VPS37A directs ESCRT recruitment for phagophore closure

2019 ◽  
Vol 218 (10) ◽  
pp. 3336-3354 ◽  
Author(s):  
Yoshinori Takahashi ◽  
Xinwen Liang ◽  
Tatsuya Hattori ◽  
Zhenyuan Tang ◽  
Haiyan He ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Growth Factor Receptor ◽  
Multivesicular Body ◽  
Regulatory Mechanisms ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Escrt Machinery ◽  
Genome Wide ◽  
A Genome ◽  
Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

scholarly journals Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

2020 ◽  
Vol 117 (41) ◽  
pp. 25293-25301
Author(s):  
Marjoke F. Debets ◽  
Omur Y. Tastan ◽  
Simon P. Wisnovsky ◽  
Stacy A. Malaker ◽  
Nikolaos Angelis ◽  
...  
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Ectopic Expression ◽  
Protein Glycosylation ◽  
Major Type ◽  
Metabolic Labeling ◽  
Uridine Diphosphate ◽  
Superresolution Microscopy ◽  
Genome Wide ◽  
A Genome

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probeN-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)–linkedN-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branchedN-acylamide side chain, GalNAzMe is not interconverted by epimerization to the correspondingN-acetylglucosamine analog by the epimeraseN-acetylgalactosamine–4-epimerase (GALE) like conventional GalNAc–based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan–specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, “bump-and-hole” (BH)–GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

scholarly journals Genome-wide identification of alternative splicing events that regulate protein transport across the secretory pathway

2019 ◽  
Vol 132 (8) ◽  
pp. jcs230201 ◽  
Author(s):  
Alexander Neumann ◽  
Magdalena Schindler ◽  
Didrik Olofsson ◽  
Ilka Wilhelmi ◽  
Annette Schürmann ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Genome Wide ◽  
Alternative Splicing Events ◽  
Regulate Protein

scholarly journals The Antisense Transcriptomes of Human Cells

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1855-1857 ◽  
Author(s):  
Yiping He ◽  
Bert Vogelstein ◽  
Victor E. Velculescu ◽  
Nickolas Papadopoulos ◽  
Kenneth W. Kinzler
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Human Cells ◽  
Antisense Transcripts ◽  
Genome Wide ◽  
Human Genes ◽  
A Genome ◽  
Dna Strand ◽  
The Relationship ◽  
Rna Transcript

Transcription in mammalian cells can be assessed at a genome-wide level, but it has been difficult to reliably determine whether individual transcripts are derived from the plus or minus strands of chromosomes. This distinction can be critical for understanding the relationship between known transcripts (sense) and the complementary antisense transcripts that may regulate them. Here, we describe a technique that can be used to (i) identify the DNA strand of origin for any particular RNA transcript, and (ii) quantify the number of sense and antisense transcripts from expressed genes at a global level. We examined five different human cell types and in each case found evidence for antisense transcripts in 2900 to 6400 human genes. The distribution of antisense transcripts was distinct from that of sense transcripts, was nonrandom across the genome, and differed among cell types. Antisense transcripts thus appear to be a pervasive feature of human cells, which suggests that they are a fundamental component of gene regulation.

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