scholarly journals A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

2007 ◽  
Vol 18 (2) ◽  
pp. 414-425 ◽  
Author(s):  
Rahel Byland ◽  
Patricia J. Vance ◽  
James A. Hoxie ◽  
Mark Marsh
Keyword(s):  
Transmembrane Protein ◽  
Viral Envelope ◽  
Membrane Expression ◽  
Dileucine Motif ◽  
Plasma Membrane Expression ◽  
Protein Gp41 ◽  
Clathrin Adaptor ◽  
Cellular Components ◽  
Specific Membrane

During the assembly of enveloped viruses viral and cellular components essential for infectious particles must colocalize at specific membrane locations. For the human and simian immunodeficiency viruses (HIV and SIV), sorting of the viral envelope proteins (Env) to assembly sites is directed by trafficking signals located in the cytoplasmic domain of the transmembrane protein gp41 (TM). A membrane proximal conserved GYxxØ motif mediates endocytosis through interaction with the clathrin adaptor AP-2. However, experiments with SIVmac239Env indicate the presence of additional signals. Here we show that a conserved C-terminal dileucine in HIVHxB2also mediates endocytosis. Biochemical and morphological assays demonstrate that the C-terminal dileucine motif mediates internalization as efficiently as the GYxxØ motif and that both must be removed to prevent Env internalization. RNAi experiments show that depletion of the clathrin adaptor AP-2 leads to increased plasma membrane expression of HIV Env and that this adaptor is required for efficient internalization mediated by both signals. The redundancy of conserved endocytosis signals and the role of the SIVmac239Env GYxxØ motif in SIV pathogenesis, suggest that these motifs have functions in addition to endocytosis, possibly related to Env delivery to the site of viral assembly and/or incorporation into budding virions.

scholarly journals CombiFlow: Combinatorial AML-specific plasma membrane expression profiles allow longitudinal tracking of clones

2021 ◽  
Author(s):  
Roos Houtsma ◽  
Nisha K. van der Meer ◽  
Kees Meijer ◽  
Linde Morsink ◽  
Shanna M. Hogeling ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Residual Disease ◽  
Expression Profiles ◽  
Clonal Evolution ◽  
Membrane Expression ◽  
Routine Diagnostics ◽  
Free Survival ◽  
Longitudinal Tracking ◽  
Plasma Membrane Expression

Acute myeloid leukemia (AML) often presents as an oligoclonal disease whereby multiple genetically distinct subclones can co-exist within patients. Differences in signaling and drug sensitivity of such subclones complicates treatment and warrants tools to identify them and track disease progression. We previously identified over 50 AML-specific plasma membrane (PM) proteins and seven of these (CD82, CD97, FLT3, IL1RAP, TIM3, CD25 and CD123) were implemented in routine diagnostics in patients with AML (n=256) and MDS (n=33). We developed a pipeline termed CombiFlow in which expression data of multiple PM markers is merged, allowing a Principle Component-based analyses to identify distinctive marker expression profiles and to generate single cell tSNE landscapes to longitudinally track clonal evolution. Positivity for one or more of the markers after 2 courses of intensive chemotherapy predicted a shorter relapse-free survival supporting a role of these markers in measurable residual disease (MRD) detection. CombiFlow also allowed the tracking of clonal evolution in paired diagnosis and relapse samples (n=12). Extending the panel to 36 AML-specific markers further refined the CombiFlow pipeline. In conclusion, CombiFlow provides a valuable tool in the diagnosis, MRD detection, clonal tracking, and the understanding of clonal heterogeneity in AML.

Bidirectional regulation of AQP2 trafficking and recycling: involvement of AQP2-S256 phosphorylation

2005 ◽  
Vol 288 (5) ◽  
pp. F930-F938 ◽  
Author(s):  
Lene N. Nejsum ◽  
Marina Zelenina ◽  
Anita Aperia ◽  
Jørgen Frøkiær ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Rat Kidney ◽  
Wild Type ◽  
Membrane Expression ◽  
Bidirectional Regulation ◽  
Plasma Membrane Expression ◽  
Intracellular Vesicles ◽  
Membrane Treatment ◽  
Microscopic Techniques

The present study examined the role of PKA and serine256 (S256) phosphorylation for AQP2 trafficking and recycling using cells transfected with wild-type AQP2 (AQP2-WT) or mutant AQP2 and high-resolution confocal microscopic techniques. In transiently transfected MDCK-C7 cells, stimulation with forskolin induced translocation of AQP2-WT to the plasma membrane. Treatment of AQP2-WT cells with the PKA inhibitor H-89 following forskolin stimulation resulted in internalization of AQP2-WT. Moreover, H-89 treatment of AQP2-S256D (mimicking constitutively phosphorylated AQP2 and hence localized to the plasma membrane) resulted in redistribution of AQP2-S256D to intracellular vesicles, even in the presence of forskolin. Both PGE2 and dopamine stimulation induced endocytosis of AQP2-WT and AQP2-S256D, respectively, in forskolin-stimulated cells. Consistent with this, dopamine in the presence of vasopressin stimulated endocytosis of AQP2 in slices of rat kidney inner medulla without substantial dephosphorylation. In conclusion, these results strongly suggest that 1) S256 phosphorylation is necessary but not sufficient for AQP2 plasma membrane expression, 2) active PKA is required for AQP2 plasma membrane expression, 3) PGE2 and dopamine induce internalization of AQP2 independently of AQP2 dephosphorylation, and 4) preceding activation of cAMP production is necessary for PGE2 and dopamine to cause AQP2 internalization.

scholarly journals Fusogenic micropeptide Myomixer is essential for satellite cell fusion and muscle regeneration

2018 ◽  
Vol 115 (15) ◽  
pp. 3864-3869 ◽  
Author(s):  
Pengpeng Bi ◽  
John R. McAnally ◽  
John M. Shelton ◽  
Efrain Sánchez-Ortiz ◽  
Rhonda Bassel-Duby ◽  
...  
Keyword(s):  
Satellite Cell ◽  
Cell Fusion ◽  
Satellite Cells ◽  
Muscle Regeneration ◽  
Transmembrane Protein ◽  
Myoblast Fusion ◽  
Conditional Knockout ◽  
Genetic Deletion ◽  
Specific Membrane

Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.

scholarly journals Analysis of Mason-Pfizer Monkey Virus Gag Domains Required for Capsid Assembly in Bacteria: Role of the N-Terminal Proline Residue of CA in Directing Particle Shape

2000 ◽  
Vol 74 (18) ◽  
pp. 8452-8459 ◽  
Author(s):  
Michaela Rumlova-Klikova ◽  
Eric Hunter ◽  
Milan V. Nermut ◽  
Iva Pichova ◽  
Tomas Ruml
Keyword(s):  
Amino Acid ◽  
Nucleocapsid Protein ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Amino Acid Residues ◽  
Membrane Expression ◽  
Specific Amino Acid ◽  
Plasma Membrane Expression ◽  
C And N

ABSTRACT Mason-Pfizer monkey virus (M-PMV) preassembles immature capsids in the cytoplasm prior to transporting them to the plasma membrane. Expression of the M-PMV Gag precursor in bacteria results in the assembly of capsids indistinguishable from those assembled in mammalian cells. We have used this system to investigate the structural requirements for the assembly of Gag precursors into procapsids. A series of C- and N-terminal deletion mutants progressively lacking each of the mature Gag domains (matrix protein [MA]-pp24/16-p12-capsid protein [CA]-nucleocapsid protein [NC]-p4) were constructed and expressed in bacteria. The results demonstrate that both the CA and the NC domains are necessary for the assembly of macromolecular arrays (sheets) but that amino acid residues at the N terminus of CA define the assembly of spherical capsids. The role of these N-terminal domains is not based on a specific amino acid sequence, since both MA-CA-NC and p12-CA-NC polyproteins efficiently assemble into capsids. Residues N terminal of CA appear to prevent a conformational change in which the N-terminal proline plays a key role, since the expression of a CA-NC protein lacking this proline results in the assembly of spherical capsids in place of the sheets assembled by the CA-NC protein.

A dileucine motif is involved in plasma membrane expression and endocytosis of rat sodium taurocholate cotransporting polypeptide (Ntcp)

2013 ◽  
Vol 305 (10) ◽  
pp. G722-G730 ◽  
Author(s):  
Claudia Stross ◽  
Stefanie Kluge ◽  
Katrin Weissenberger ◽  
Elisabeth Winands ◽  
Dieter Häussinger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sodium Taurocholate ◽  
Dual Function ◽  
Intracellular Loop ◽  
Membrane Expression ◽  
Dileucine Motif ◽  
Plasma Membrane Expression ◽  
Third Intracellular Loop ◽  
Parenchymal Cells ◽  
Pkc Activation

The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells, and PKC-mediated endocytosis was shown to regulate the number of Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence, and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by ∼30%. Endocytosis of Ntcp was clathrin dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two of five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.

scholarly journals Role of the Dileucine Motif in Nef-Induced Trimerization of the ARF1:AP-1 Clathrin Adaptor Complex

2018 ◽  
Vol 114 (3) ◽  
pp. 570a
Author(s):  
Cosmo Z. Buffalo ◽  
Kyle L. Morris ◽  
Xuefeng Ren ◽  
James H. Hurley
Keyword(s):  
Dileucine Motif ◽  
Clathrin Adaptor

The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets

2007 ◽  
Vol 292 (5) ◽  
pp. C1690-C1700 ◽  
Author(s):  
Neelakshi R. Jog ◽  
Madhavi J. Rane ◽  
George Lominadze ◽  
Gregory C. Luerman ◽  
Richard A. Ward ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Immunoblot Analysis ◽  
Maximal Response ◽  
Initial Increase ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Latrunculin A ◽  
Specific Granules

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED50of 15, 31, and 28 nM, respectively, with maximal response at 10−7M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC50for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis.

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