scholarly journals An application of fusion fluorescent proteins in the pharmacological study of intracellular protein trafficking.

2000 ◽  
Vol 116 (1) ◽  
pp. 36-42
Author(s):  
Toshimasa YOSHIOKA ◽  
Fujiko TSUKAHARA
Keyword(s):  
Protein Trafficking ◽  
Fluorescent Proteins ◽  
Pharmacological Study ◽  
Intracellular Protein

scholarly journals The Golgi-associated retrograde protein (GARP) complex plays an essential role in the maintenance of the Golgi glycosylation machinery

2020 ◽  
Author(s):  
Amrita Khakurel ◽  
Tetyana Kudlyk ◽  
Juan S. Bonifacino ◽  
Vladimir V. Lupashin
Keyword(s):  
Steady State ◽  
Golgi Apparatus ◽  
Protein Trafficking ◽  
Essential Role ◽  
Human Cell Lines ◽  
Critical Component ◽  
Intracellular Protein ◽  
Vesicle Recycling ◽  
The Stability ◽  
Garp Complex

AbstractThe Golgi apparatus is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and vesicle recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits is detrimental to N- and O-glycosylation, and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-KO cells exhibit impaired retention of glycosylation enzymes in the Golgi. Indeed, a RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi but instead is missorted to the endolysosomal compartment. We propose that the endosomal compartment is part of the trafficking itinerary of Golgi enzymes and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery.

scholarly journals Identification of SPPL2a Inhibitors by Multiparametric Analysis of a High-Content Ultra-High-Throughput Screen

2017 ◽  
Vol 22 (9) ◽  
pp. 1106-1119 ◽  
Author(s):  
Xian Zhang ◽  
Marjo Götte ◽  
Yvonne Ibig-Rehm ◽  
Ansgar Schuffenhauer ◽  
Marion Kamke ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Trafficking ◽  
Drug Target ◽  
Nuclear Translocation ◽  
State Of The Art ◽  
Screening Strategy ◽  
Analysis Method ◽  
Intracellular Protein ◽  
Multiparametric Analysis ◽  
Cellular Phenotypes

The intramembrane protease signal peptide peptidase-like 2a (SPPL2a) is a potential drug target for the treatment of autoimmune diseases due to an essential role in B cells and dendritic cells. To screen a library of 1.4 million compounds for inhibitors of SPPL2a, we developed an imaging assay detecting nuclear translocation of the proteolytically released cytosolic substrate fragment. The state-of-the-art hit calling approach based on nuclear translocation resulted in numerous false-positive hits, mainly interrupting intracellular protein trafficking. To filter the false positives, we extracted 340 image-based readouts and developed a novel multiparametric analysis method that successfully triaged the primary hit list. The identified scaffolds were validated by demonstrating activity on endogenous SPPL2a and substrate CD74/p8 in B cells. The multiparametric analysis discovered diverse cellular phenotypes and provided profiles for the whole library. The principle of the presented imaging assay, the screening strategy, and multiparametric analysis are potentially applicable in future screening campaigns.

Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2

2007 ◽  
Vol 2 (8) ◽  
pp. 2024-2032 ◽  
Author(s):  
Dmitriy M Chudakov ◽  
Sergey Lukyanov ◽  
Konstantin A Lukyanov
Keyword(s):  
Fluorescent Proteins ◽  
Intracellular Protein

scholarly journals Formin 1 Regulates Microtubule and F-Actin Organization to Support Spermatid Transport During Spermatogenesis in the Rat Testis

Endocrinology ◽  
2016 ◽  
Vol 157 (7) ◽  
pp. 2894-2908 ◽  
Author(s):  
Nan Li ◽  
Dolores D. Mruk ◽  
Elizabeth I. Tang ◽  
Will M. Lee ◽  
Chris K. C. Wong ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Protein Trafficking ◽  
Cell Shape ◽  
Binding Protein ◽  
Cell Movement ◽  
Seminiferous Epithelium ◽  
Intracellular Protein ◽  
Down Regulation ◽  
Actin Organization ◽  
Support Cell

Formin 1 confers actin nucleation by generating long stretches of actin microfilaments to support cell movement, cell shape, and intracellular protein trafficking. Formin 1 is likely involved in microtubule (MT) dynamics due to the presence of a MT binding domain near its N terminus. Here, formin 1 was shown to structurally interact with α-tubulin, the building block of MT, and also end-binding protein 1 (a MT plus [+]-end-binding protein that stabilizes MT) in the testis. Knockdown of formin 1 in Sertoli cells with an established tight junction barrier was found to induce down-regulation of detyrosinated MT (a stabilized form of MT), and disorganization of MTs, in which MTs were retracted from the cell cortical zone, mediated through a loss of MT polymerization and down-regulation of Akt1/2 signaling kinase. An efficient knockdown of formin 1 in the testis reduced the number of track-like structures conferred by MTs and F-actin considerably, causing defects in spermatid and phagosome transport across the seminiferous epithelium. In summary, formin1 maintains MT and F-actin track-like structures to support spermatid and phagosome transport across the seminiferous epithelium during spermatogenesis.

scholarly journals Control of Golgi Morphology and Function by Sed5 t-SNARE Phosphorylation

2005 ◽  
Vol 16 (10) ◽  
pp. 4918-4930 ◽  
Author(s):  
Adina Weinberger ◽  
Faustin Kamena ◽  
Rachel Kama ◽  
Anne Spang ◽  
Jeffrey E. Gerst
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Trafficking ◽  
Retrograde Transport ◽  
Cell Types ◽  
Intracellular Protein ◽  
Inhibition Of Growth ◽  
Golgi Transport ◽  
Transport Vesicles ◽  
And Function ◽  
Kinase A

Previously, we demonstrated that the phosphorylation of t-SNAREs by protein kinase A (PKA) affects their ability to participate in SNARE complexes and to confer endocytosis and exocytosis in yeast. Here, we show that the presumed phosphorylation of a conserved membrane-proximal PKA consensus site (serine-317) in the Sed5 t-SNARE regulates endoplasmic reticulum (ER)-Golgi transport, as well as Golgi morphology. Sed5 is a phosphoprotein, and both alanine and aspartate substitutions in serine-317 directly affect intracellular protein trafficking. The aspartate substitution results in elaboration of the ER, defects in Golgi-ER retrograde transport, an accumulation of small transport vesicles, and the inhibition of growth of most cell types. In contrast, the alanine substitution has no deleterious effects upon transport and growth, but results in ordering of the Golgi into a structure reminiscent of mammalian apparatus. This structure seems to require the recycling of Sed5, because it was found not to occur in sec21-2 cells that are defective in retrograde transport. Thus, a cycle of Sed5 phosphorylation and dephosphorylation is required for normal t-SNARE function and may choreograph Golgi ordering and dispersal.

scholarly journals Proteomics of protein trafficking by in vivo tissue-specific labeling

2020 ◽  
Author(s):  
Ilia A. Droujinine ◽  
Dan Wang ◽  
Yanhui Hu ◽  
Namrata D. Udeshi ◽  
Luye Mu ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Large Scale ◽  
Fat Body ◽  
Secreted Proteins ◽  
Intracellular Protein ◽  
Quantitative Mass Spectrometry ◽  
Subcellular Compartment ◽  
Human Orthologs ◽  
Identification And Characterization

AbstractSecreted interorgan communication factors encode key regulators of homeostasis. However, long-standing questions surround their origins/destinations, mechanisms of interactions, and the number of proteins involved. Progress has been hindered by the lack of methodologies for these factors’ large-scale identification and characterization, as conventional approaches cannot identify low-abundance factors and the origins and destinations of secreted proteins. We established an in vivo platform to investigate secreted protein trafficking between organs proteome-wide, whereby engineered promiscuous biotin ligase BirA*G3 (a relative of TurboID) biotinylates all proteins in a subcellular compartment of one tissue, and biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Using this platform, we identified 51 putative muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, of which 60-70% have human orthologs. We demonstrate, in particular, that conserved fat body-derived novel interorgan communication factors CG31326, CG2145, and CG4332 promote muscle activity. Our results indicate that the communication network of secreted proteins is vast, and we identified systemic functions for a number of these factors. This approach is widely applicable to studies in interorgan, local and intracellular protein trafficking networks, non-conventional secretion, and to mammalian systems, under healthy or diseased states.One Sentence SummaryWe developed an in vivo platform to investigate protein trafficking between organs proteome-wide, provide a resource for interorgan communication factors, and determined conserved adipokines that affect muscles.

scholarly journals The Golgi-associated retrograde protein (GARP) complex plays an essential role in the maintenance of the Golgi glycosylation machinery

2021 ◽  
pp. mbc.E21-04-0169
Author(s):  
Amrita Khakurel ◽  
Tetyana Kudlyk ◽  
Juan S. Bonifacino ◽  
Vladimir V. Lupashin
Keyword(s):  
Steady State ◽  
Golgi Complex ◽  
Protein Trafficking ◽  
Essential Role ◽  
Human Cell Lines ◽  
Critical Component ◽  
Intracellular Protein ◽  
The Stability ◽  
Endosomal System ◽  
Garp Complex

The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans, and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-KO cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text]

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