scholarly journals Probing the Subcellular Distribution of Phosphatidylinositol Reveals a Surprising Lack at the Plasma Membrane

2019 ◽  
Author(s):  
James P. Zewe ◽  
April Miller ◽  
Sahana Sangappa ◽  
Rachel C. Wills ◽  
Brady D. Goulden ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Subcellular Distribution ◽  
Eukaryotic Cells ◽  
Intracellular Membrane ◽  
Membrane Function ◽  
Membrane Compartments ◽  
Health And Disease ◽  
Structural Lipid ◽  
And Function

AbstractThe polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids’ role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn have been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we have used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared to the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.SummaryZewe et al develop approaches to map the subcellular distribution of the major phospholipid, phosphatidylinositol (PI), revealing that the lipid is present in most membranes except for plasma membrane, where it is mainly found as PI4P and PI(4,5)P2.

scholarly journals Probing the subcellular distribution of phosphatidylinositol reveals a surprising lack at the plasma membrane

2020 ◽  
Vol 219 (3) ◽  
Author(s):  
James P. Zewe ◽  
April M. Miller ◽  
Sahana Sangappa ◽  
Rachel C. Wills ◽  
Brady D. Goulden ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Subcellular Distribution ◽  
Eukaryotic Cells ◽  
Intracellular Membrane ◽  
Membrane Function ◽  
Membrane Compartments ◽  
Health And Disease ◽  
Structural Lipid ◽  
And Function

The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids’ role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.

scholarly journals Impact of Membrane Lipids on UapA and AzgA Transporter Subcellular Localization and Activity in Aspergillus nidulans

2021 ◽  
Vol 7 (7) ◽  
pp. 514
Author(s):  
Mariangela Dionysopoulou ◽  
George Diallinas
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Aspergillus Nidulans ◽  
Membrane Lipids ◽  
Membrane Lipid ◽  
Lipid Biosynthesis ◽  
Transport Kinetics ◽  
Negative Effect ◽  
And Function

Recent biochemical and biophysical evidence have established that membrane lipids, namely phospholipids, sphingolipids and sterols, are critical for the function of eukaryotic plasma membrane transporters. Here, we study the effect of selected membrane lipid biosynthesis mutations and of the ergosterol-related antifungal itraconazole on the subcellular localization, stability and transport kinetics of two well-studied purine transporters, UapA and AzgA, in Aspergillus nidulans. We show that genetic reduction in biosynthesis of ergosterol, sphingolipids or phosphoinositides arrest A. nidulans growth after germling formation, but solely blocks in early steps of ergosterol (Erg11) or sphingolipid (BasA) synthesis have a negative effect on plasma membrane (PM) localization and stability of transporters before growth arrest. Surprisingly, the fraction of UapA or AzgA that reaches the PM in lipid biosynthesis mutants is shown to conserve normal apparent transport kinetics. We further show that turnover of UapA, which is the transporter mostly sensitive to membrane lipid content modification, occurs during its trafficking and by enhanced endocytosis, and is partly dependent on autophagy and Hect-type HulARsp5 ubiquitination. Our results point out that the role of specific membrane lipids on transporter biogenesis and function in vivo is complex, combinatorial and transporter-dependent.

Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 4006-4014 ◽  
Author(s):  
Dian Feng ◽  
Katharine Crane ◽  
Nataliya Rozenvayn ◽  
Ann M. Dvorak ◽  
Robert Flaumenhaft
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Molecular Mechanisms ◽  
Protein Composition ◽  
Immunoblot Analysis ◽  
Snare Proteins ◽  
Ultrastructural Studies ◽  
Streptolysin O ◽  
Membrane Compartments ◽  
Granule Secretion

Morphologic studies have demonstrated a process by which α-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for α-granule secretion. These observations raise the possibility that morphologic features of α-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins—human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca++-induced α-granule secretion by approximately 50%. Inhibition of α-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of α-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of α-granule secretion that include homotypic α-granule fusion and the fusion of α-granules with the open canalicular system and plasma membrane.

Initial entry of IRAP into the insulin-responsive storage compartment occurs prior to basal or insulin-stimulated plasma membrane recycling

2005 ◽  
Vol 289 (5) ◽  
pp. E746-E752 ◽  
Author(s):  
Gang Liu ◽  
June Chunqiu Hou ◽  
Robert T. Watson ◽  
Jeffrey E. Pessin
Keyword(s):  
Plasma Membrane ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Intracellular Membrane ◽  
Receptor Endocytosis ◽  
Membrane Compartments ◽  
Insulin Responsiveness ◽  
Green Fluorescent ◽  
Syntaxin 3

To examine the acquisition of insulin sensitivity after the initial biosynthesis of the insulin-responsive aminopeptidase (IRAP), 3T3-L1 adipocytes were transfected with an enhanced green fluorescent protein-IRAP (EGFP-IRAP) fusion protein. In the absence of insulin, IRAP was rapidly localized (1–3 h) to secretory membranes and retained in these intracellular membrane compartments with little accumulation at the plasma membrane. However, insulin was unable to induce translocation to the plasma membrane until 6–9 h after biosynthesis. This was in marked contrast to another type II membrane protein (syntaxin 3) that rapidly defaulted to the plasma membrane 3 h after expression. In parallel with the time-dependent acquisition of insulin responsiveness, the newly synthesized IRAP protein converted from a brefeldin A-sensitive to a brefeldin A-insensitive state. The initial trafficking of IRAP to the insulin-responsive compartment was independent of plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis but had no effect on the insulin-stimulated translocation of the newly synthesized IRAP protein.

Cellular microcompartments constitute general suborganellar functional units in cells

2013 ◽  
Vol 394 (2) ◽  
pp. 151-161 ◽  
Author(s):  
Joost C.M. Holthuis ◽  
Christian Ungermann
Keyword(s):  
Plasma Membrane ◽  
Cell Signaling ◽  
Functional Unit ◽  
Redox Status ◽  
Eukaryotic Cells ◽  
Enzymatic Reactions ◽  
Ion Composition ◽  
Cellular Dynamics ◽  
Remarkable Degree ◽  
And Function

Abstract All cells are compartmentalized to facilitate enzymatic reactions or cellular dynamics. In eukaryotic cells, organelles differ in their protein/lipid repertoire, luminal ion composition, pH, and redox status. In addition, organelles contain specialized subcompartments even within the same membrane or within its lumen. Moreover, the bacterial plasma membrane reveals a remarkable degree of organization, which is recapitulated in eukaryotic cells and often linked to cell signaling. Finally, protein-based compartments are also known in the bacterial and eukaryotic cytosol. As the organizing principle of such cellular subcompartments is likely similar, previous definitions like rafts, microdomains, and all kinds of ‘-somes’ fall short as a general denominator to describe such suborganellar structures. Within this review, we will introduce the term cellular microcompartment as a general suborganellar functional unit and discuss its relevance to understand subcellular organization and function.

Microcompartments within the yeast plasma membrane

2013 ◽  
Vol 394 (2) ◽  
pp. 189-202 ◽  
Author(s):  
Hans Merzendorfer ◽  
Jürgen J. Heinisch
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Model Organism ◽  
Dynamic Interaction ◽  
Eukaryotic Cells ◽  
Classical Concept ◽  
Yeast Plasma Membrane ◽  
Bud Neck ◽  
Microscopic Studies ◽  
Membrane Compartments

Abstract Recent research in cell biology makes it increasingly clear that the classical concept of compartmentation of eukaryotic cells into different organelles performing distinct functions has to be extended by microcompartmentation, i.e., the dynamic interaction of proteins, sugars, and lipids at a suborganellar level, which contributes significantly to a proper physiology. As different membrane compartments (MCs) have been described in the yeast plasma membrane, such as those defined by Can1 and Pma1 (MCCs and MCPs), Saccharomyces cerevisiae can serve as a model organism, which is amenable to genetic, biochemical, and microscopic studies. In this review, we compare the specialized microcompartment of the yeast bud neck with other plasma membrane substructures, focusing on eisosomes, cell wall integrity-sensing units, and chitin-synthesizing complexes. Together, they ensure a proper cell division at the end of mitosis, an intricately regulated process, which is essential for the survival and proliferation not only of fungal, but of all eukaryotic cells.

scholarly journals The Subcellular Localization of an Aquaporin-2 Tetramer Depends on the Stoichiometry of Phosphorylated and Nonphosphorylated Monomers

2000 ◽  
Vol 151 (4) ◽  
pp. 919-930 ◽  
Author(s):  
E.J. Kamsteeg ◽  
I. Heijnen ◽  
C.H. van Os ◽  
P.M.T. Deen
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Subcellular Localization ◽  
Water Channel ◽  
Apical Plasma Membrane ◽  
Wild Type ◽  
Principal Cells ◽  
Intracellular Vesicles ◽  
And Function ◽  
Fine Tune

In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non–p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes.

scholarly journals Schizosaccharomyces pombe Sst4p, a Conserved Vps27/Hrs Homolog, Functions Downstream of Phosphatidylinositol 3-Kinase Pik3p To Mediate Proper Spore Formation

2007 ◽  
Vol 6 (12) ◽  
pp. 2343-2353 ◽  
Author(s):  
Masayuki Onishi ◽  
Michihiro Iida ◽  
Takako Koga ◽  
Sadayuki Yamada ◽  
Aiko Hirata ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fluorescent Protein ◽  
Developmental Process ◽  
Intracellular Membrane ◽  
Phosphatidylinositol 3 Kinase ◽  
Fyve Domain ◽  
Membrane Compartments ◽  
And Function ◽  
Phosphatidylinositol 3

ABSTRACT Sporulation of the fission yeast Schizosaccharomyces pombe is a developmental process that generates gametes and that includes the formation of spore envelope precursors called the forespore membranes. Assembly and development of forespore membranes require vesicular trafficking from other intracellular membrane compartments. We have shown that phosphatidylinositol 3-kinase (PtdIns 3-kinase) is required for efficient and proper development of forespore membranes. The role of a FYVE domain protein, Sst4p, a homolog of Vps27p/Hrs, as a downstream factor for PtdIns 3-kinase in sporulation was investigated. sst4Δ asci formed spores with oval-shaped morphology and with reduced viability compared to that of the wild-type spores. The extension of forespore membranes was inefficient, and bubble-like structures emerged from the leading edges of the forespore membranes. Sst4p localization was examined using fluorescent protein fusions and was found to be adjacent to the forespore membranes during sporulation. The localization and function of Sst4p were dependent on its FYVE domain and on PtdIns 3-kinase. Sst4p colocalized and interacted with Hse1p, a homolog of Saccharomyces cerevisiae Hse1p and of mammalian STAM. Mutations in all three UIM domains of the Sst4p/Hse1p complex resulted in formation of spores with abnormal morphology. These results suggest that Sst4p is a downstream factor of PtdIns 3-kinase and functions in forespore membrane formation.

scholarly journals Structure and Function of the Plasma Membrane

1968 ◽  
Vol 52 (1) ◽  
pp. 257-278 ◽  
Author(s):  
Edward D. Korn
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Current Knowledge ◽  
Structure And Function ◽  
Attractive Alternative ◽  
Membrane Function ◽  
Classical Models ◽  
And Function ◽  
Direct Attack ◽  
Membrane Biosynthesis

The paucimolecular unit membrane model of the structure of the plasma membrane is critically reviewed in relation to current knowledge of the chemical and enzymatic composition of isolated plasma membranes, the properties of phospholipids, the chemistry of fixation for electron microscopy, the conformation of membrane proteins, the nature of the lipid-protein bonds in membranes, and possible mechanisms of transmembrane transport and membrane biosynthesis. It is concluded that the classical models, although not disproven, are not well supported by, and are difficult to reconcile with, the data now available. On the other hand, although a model based on lipoprotein subunits is, from a biochemical perspective, an attractive alternative, it too is far from proven. Many of the questions may be resolved by studies of membrane function and membrane biosynthesis rather than by a direct attack on membrane structure.

Prospective of Ras signaling in stem cells

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Koushik Chakrabarty ◽  
Rolf Heumann
Keyword(s):  
Stem Cells ◽  
Biological Process ◽  
Large Family ◽  
Eukaryotic Cells ◽  
Signal Transduction Pathways ◽  
Ras Signaling ◽  
Intracellular Membrane ◽  
Predominant Role ◽  
Ras Signaling Pathway ◽  
Membrane Compartments

Abstract The Ras signaling pathway plays a predominant role during development and controls diverse biological process in all eukaryotic cells. It is a member of the large family of GTPases proteins that binds and hydrolyzes GTP. Ras is a lipid-anchored protein on the intracellular membrane compartments, and cycles between inactive GDP-bound and the signaling competent GTP-bound conformation. Studies have demonstrated Ras to be a central regulator in signal transduction pathways responding to diverse extracellular and intracellular stimuli. Much progress has been made towards delineating specific genes involved in the process of pluripotency and differentiation of stem cells. Here, we discuss recent aspects of Ras signaling pathways in mediating stem cell properties.

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