scholarly journals The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis

Blood ◽  
2012 ◽  
Vol 120 (25) ◽  
pp. 5032-5040 ◽  
Author(s):  
Denisa Urban ◽  
Ling Li ◽  
Hilary Christensen ◽  
Fred G. Pluthero ◽  
Shao Zun Chen ◽  
...  
Keyword(s):  
Immunofluorescence Microscopy ◽  
Binding Protein ◽  
Yeast Two Hybrid ◽  
Binding Partner ◽  
Late Endosomes ◽  
Arc Syndrome ◽  
Membrane Bound ◽  
Golgi Network ◽  
Two Hybrid

Abstract Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis.

scholarly journals The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

1998 ◽  
Vol 141 (7) ◽  
pp. 1515-1527 ◽  
Author(s):  
Ping Lin ◽  
Helen Le-Niculescu ◽  
Robert Hofmeister ◽  
J. Michael McCaffery ◽  
Mingjie Jin ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Yeast Two Hybrid ◽  
Golgi Membranes ◽  
Yeast Two Hybrid System ◽  
Golgi Region ◽  
Ef Hand ◽  
Golgi Network ◽  
Two Hybrid

We have identified CALNUC, an EF-hand, Ca2+-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gαi3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gαi3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca2+-binding protein detected by 45Ca2+-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.

Selection and characterization of large collections of peptide aptamers through optimized yeast two-hybrid procedures

2006 ◽  
Vol 1 (3) ◽  
pp. 1066-1091 ◽  
Author(s):  
Marc B T Bickle ◽  
Eric Dusserre ◽  
Olivier Moncorgé ◽  
Hélène Bottin ◽  
Pierre Colas
Keyword(s):  
Yeast Two Hybrid ◽  
Hybrid Procedures ◽  
Peptide Aptamers ◽  
Two Hybrid

scholarly journals A novel yeast two-hybrid approach to identify CDPK substrates: Characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein1

FEBS Letters ◽  
2006 ◽  
Vol 580 (3) ◽  
pp. 904-911 ◽  
Author(s):  
Miguel A. Rodriguez Milla ◽  
Yuichi Uno ◽  
Ing-Feng Chang ◽  
Jared Townsend ◽  
Eileen A. Maher ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Hybrid Approach ◽  
Yeast Two Hybrid ◽  
Two Hybrid

scholarly journals Involvement of Actinin-4 in the Recruitment of JRAB/MICAL-L2 to Cell-Cell Junctions and the Formation of Functional Tight Junctions

2008 ◽  
Vol 28 (10) ◽  
pp. 3324-3335 ◽  
Author(s):  
Hiroyoshi Nakatsuji ◽  
Noriyuki Nishimura ◽  
Rie Yamamura ◽  
Hiro-omi Kanayama ◽  
Takuya Sasaki
Keyword(s):  
Tight Junctions ◽  
Binding Protein ◽  
Cell Junctions ◽  
Actin Binding ◽  
Deletion Mutants ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Interfering Rna ◽  
Two Hybrid ◽  
Cell Cell

ABSTRACT Tight junctions (TJs) are cell-cell adhesive structures that undergo continuous remodeling. We previously demonstrated that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) localized at TJs and mediated the endocytic recycling of the integral TJ protein occludin and the formation of functional TJs. Here, we investigated how JRAB/MICAL-L2 was targeted to TJs. Using a series of deletion mutants, we found the plasma membrane (PM)-targeting domain within JRAB/MICAL-L2. We then identified actinin-4, which was originally isolated as an actin-binding protein associated with cell motility and cancer invasion/metastasis, as a binding protein for the PM-targeting domain of JRAB/MICAL-L2, using a yeast two-hybrid system. Actinin-4 was colocalized with JRAB/MICAL-L2 at cell-cell junctions and linked JRAB/MICAL-L2 to F-actin. Although actinin-4 bound to JRAB/MICAL-L2 without Rab13, the actinin-4-JRAB/MICAL-L2 interaction was enhanced by Rab13 activation. Depletion of actinin-4 by using small interfering RNA inhibited the recruitment of occludin to TJs during the Ca2+ switch. During the epithelial polarization after replating, JRAB/MICAL-L2 was recruited from the cytosol to cell-cell junctions. This JRAB/MICAL-L2 recruitment as well as the formation of functional TJs was delayed in actinin-4-depleted cells. These results indicate that actinin-4 is involved in recruiting JRAB/MICAL-L2 to cell-cell junctions and forming functional TJs.

scholarly journals Tubulin Folding Cofactor TBCB is a Target of the Salmonella Effector Protein SseK1

2020 ◽  
Vol 21 (9) ◽  
pp. 3193 ◽  
Author(s):  
Juan Luis Araujo-Garrido ◽  
Fernando Baisón-Olmo ◽  
Joaquín Bernal-Bayard ◽  
Francisco Romero ◽  
Francisco Ramos-Morales
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Yeast Two Hybrid ◽  
Microtubule Network ◽  
Binding Partner ◽  
Tubulin Binding ◽  
Serovar Typhimurium ◽  
Novel Target ◽  
Two Hybrid

Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.

Identification of a VPS33B-Binding Protein That Facilitates Alpha Granule Formation In Human Megakaryocytes and Platelets.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1444-1444
Author(s):  
Denisa Urban ◽  
Ling Li ◽  
James Wasmuth ◽  
Hilary Christensen ◽  
John Parkinson ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Human Platelets ◽  
Multiprotein Complex ◽  
Yeast Two Hybrid ◽  
Granule Formation ◽  
Reading Frame ◽  
Binding Partner ◽  
Library Screen ◽  
Two Hybrid ◽  
Granule Release

Abstract Abstract 1444 Human platelets contain α-granules, dense (δ-) granules and lysosomes that release their contents upon platelet activation. Platelet granule release is important for hemostasis, since patients with inherited granule defects have bleeding problems. α-granules are absent in the gray platelet and ARC syndromes, while deficient δ-granules are observed in isolation, in combination with α-granule deficiency, or as part of a syndrome in the Hermansky-Pudlak, Chediak-Higashi and Griscelli syndromes. The biogenesis of α-granules is poorly understood. Our laboratory has identified VPS33B as a central player in the formation of platelet α-granules. VPS33B has yet to be characterized in detail, however, its homolog VPS33A is known to be part of a multiprotein complex involved intracellular vesicle trafficking. Studies in our laboratory suggest that VPS33B is also part of a multiprotein complex. We performed a yeast two-hybrid library screen with VPS33B as bait and found another member of the complex: the unidentified gene product of chromosome 14 open reading frame 133 (C14orf133). Sequence analysis indicated this to be human VPS16B. Our studies show that VPS16B specifically binds to VPS33B but not its homologue, VPS33A. Furthermore, we show that VPS33B forms a distinct complex from that of its homologue VPS33A. VPS16B was found to co-localize with trans-Golgi, late endosome and α-granule markers in megakaryocytic Dami cells. Ongoing studies suggest that knockdown of VPS16B affects α-granule formation. We conclude that VPS16B, much like its binding partner VPS33B, plays a crucial role in megakaryocyte and platelet α-granule biogenesis. Disclosures: No relevant conflicts of interest to declare.

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