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 Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli

2018 ◽  
Author(s):  
Sanduo Zheng ◽  
Lok-To Sham ◽  
Frederick A. Rubino ◽  
Kelly Brock ◽  
William P. Robins ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Biology ◽  
Model Organism ◽  
Structural Data ◽  
Outer Face ◽  
Antibiotic Development ◽  
Cell Wall Assembly ◽  
Lipid Ii

AbstractThe peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein prior to its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data are only available for MurJ from the extremophile Thermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the Gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Finally, through the use of sequence co-evolution analysis we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

scholarly journals Akt kinases are required for efficient feeding by macropinocytosis in Dictyostelium

2018 ◽  
Author(s):  
Thomas D. Williams ◽  
Sew-Yeu Peak-Chew ◽  
Peggy Paschke ◽  
Robert R. Kay
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Biology ◽  
Large Scale ◽  
Model Organism ◽  
Dependent Manner ◽  
Fluid Uptake ◽  
Small G Protein ◽  
Knockout Mutants ◽  
Summary Statement

AbstractMacropinocytosis is an actin-driven process of large-scale, non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum. In Dictyostelium, macropinocytic cups are organised by ‘macropinocytic patches’ in the plasma membrane. These contain activated Ras, Rac and PI(3,4,5)P3 and direct actin polymerisation to their periphery. Here, we show that a classical (PkbA) and a variant (PkbR1) Akt protein kinase acting downstream of PI(3,4,5)P3 are together are near-essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Akt targets identified by phosphoproteomics were highly enriched in small G-protein regulators, including the RhoGAP GacG. GacG knockout mutants make few macropinosomes but instead redeploy their cytoskeleton from macropinocytosis to motility, moving rapidly but taking up little fluid. The function of Akt in cell feeding through control of macropinosome size has implications for cancer cell biology.Summary statementDictyostelium amoebae feed by macropinocytosis in a PIP3 dependent manner. In the absence of PI3-kinases or the downstream Akt protein kinases, cells have smaller macropinosomes and nearly abolished fluid uptake.

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 Structure and mutagenic analysis of the lipid II flippase MurJ fromEscherichia coli

2018 ◽  
Vol 115 (26) ◽  
pp. 6709-6714 ◽  
Author(s):  
Sanduo Zheng ◽  
Lok-To Sham ◽  
Frederick A. Rubino ◽  
Kelly P. Brock ◽  
William P. Robins ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Biology ◽  
Model Organism ◽  
Structural Data ◽  
Outer Face ◽  
Antibiotic Development ◽  
Cell Wall Assembly ◽  
Lipid Ii ◽  
Peptidoglycan Cell Wall

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophileThermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organismEscherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability ofE. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

scholarly journals Membrane association but not identity is required for LRRK2 activation and phosphorylation of Rab GTPases

2019 ◽  
Vol 218 (12) ◽  
pp. 4157-4170 ◽  
Author(s):  
Rachel C. Gomez ◽  
Paulina Wawro ◽  
Pawel Lis ◽  
Dario R. Alessi ◽  
Suzanne R. Pfeffer
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Family Members ◽  
Membrane Association ◽  
Rab Gtpases ◽  
Membrane Compartments ◽  
Membrane Bound ◽  
Lrrk2 Kinase ◽  
Familial Parkinson’S Disease ◽  
Distinct Membrane

LRRK2 kinase mutations cause familial Parkinson’s disease and increased phosphorylation of a subset of Rab GTPases. Rab29 recruits LRRK2 to the trans-Golgi and activates it there, yet some of LRRK2’s major Rab substrates are not on the Golgi. We sought to characterize the cell biology of LRRK2 activation. Unlike other Rab family members, we show that Rab29 binds nucleotide weakly, is poorly prenylated, and is not bound to GDI in the cytosol; nevertheless, Rab29 only activates LRRK2 when it is membrane bound and GTP bound. Mitochondrially anchored, GTP-bound Rab29 is both a LRRK2 substrate and activator, and it drives accumulation of active LRRK2 and phosphorylated Rab10 on mitochondria. Importantly, mitochondrially anchored LRRK2 is much less capable of phosphorylating plasma membrane–anchored Rab10 than soluble LRRK2. These data support a model in which LRRK2 associates with and dissociates from distinct membrane compartments to phosphorylate Rab substrates; if anchored, LRRK2 can modify misdelivered Rab substrates that then become trapped there because GDI cannot retrieve them.

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.

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