#membrane protein

Basics of Cell Biology

Which of the following proteins is an integral membrane protein?

Integral membrane proteins are defined by hydrophobic transmembrane segments, typically 20-25 residues forming alpha helix crossing hydrocarbon core stabilized by van der Waals and hydrogen bonding, or beta-barrel of multiple antiparallel strands as in VDAC. Extraction requires detergent disrupting bilayer rather than high salt. Aquaporin family represents classic multipass integral protein: tetramer in plasma membrane where each monomer with six transmembrane helices forms hourglass pore lined by NPA motifs Asn-Pro-Ala that orient water molecules in single file and aromatic-arginine constriction selecting against protons, facilitating rapid osmotic water equilibration essential in kidney proximal tubule, collecting duct regulated by vasopressin, and astrocyte edema control. Spectrin is peripheral cytoskeletal tetramer binding actin via adducin and band 3 through ankyrin on inner leaflet, easily eluted with low ionic strength. Histone octamer wraps DNA in nucleus, tubulin forms cytosolic microtubules of 25 nm. Thus aquaporin exemplifies true transmembrane embedding with functional pore within bilayer, while others are soluble or peripheral.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 10: Integral Membrane Proteins, Aquaporin.

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Plasma Membrane Proteins

Which protein functions as a water channel and facilitates rapid movement of water across membranes?

Water permeability of plasma membranes exceeds lipid diffusion requiring dedicated proteinaceous pores. Aquaporin family comprising thirteen human isoforms forms homotetramers in kidney proximal tubules, red cells, and brain astrocytes each monomer about twenty eight kilodaltons six transmembrane helices arrangement N-terminus cytosolic with two half-helices HB and HE entering membrane containing invariant NPA motifs meeting at center forming aqueous pathway hourglass shape. Extracellular vestibule leads to aromatic arginine constriction formed by Arg195 His180 Phe56 limiting diameter to two point eight angstroms permitting single file waters hydrogen bonded to carbonyl oxygens and asparagine side chains while excluding hydronium via electrostatic barrier and interrupting Grotthuss proton wire. Transport occurs passive facilitated diffusion rate up to three billion molecules per second per channel driven by osmotic gradient without conformational change measured by stopped-flow light scattering. Band 3 performs anion exchange, GPCR transduces signals, spectrin provides elasticity, establishing aquaporin uniquely as water channel with dual NPA filter ensuring high selectivity.

Ref: Agre et al., Aquaporin Water Channels – Structure and Function, Nobel Lecture 2003.

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Plasma Membrane Proteins

Which type of membrane protein is covalently linked to a lipid moiety, anchoring it to the membrane?

Lipid linkage expands repertoire of membrane association beyond hydrophobic transmembrane spans enabling reversible targeting. Four major classes documented: N-myristoylation fourteen-carbon saturated fatty acid amide linked to N-terminal glycine after methionine removal catalyzed by N-myristoyltransferase cotranslationally; S-palmitoylation sixteen-carbon palmitate thioester linked to cysteine catalyzed by DHHC palmitoyl acyltransferases reversible by thioesterases controlling trafficking; prenylation fifteen-carbon farnesyl or twenty-carbon geranylgeranyl thioether linked to C-terminal CAAX cysteine by farnesyltransferase and geranylgeranyltransferases followed by proteolysis by RCE1 and carboxyl methylation by ICMT; GPI anchoring where preassembled glycolipid comprising ethanolamine phosphate oligosaccharide glucosamine mannose inositol diacylglycerol attached to C-terminal cleavage site via transamidase embedding in outer leaflet. These moieties embed in one leaflet providing raft affinity, polarized sorting and assembly of signalosomes for Ras Rab Src G-alpha and alkaline phosphatase families distinct from multipass integral proteins like channels that cross bilayer via peptide helices. Each modification uses distinct metabolite donors myristoyl-CoA palmitoyl-CoA farnesyl diphosphate and preassembled GPI precursor dictating substrate specificity and cellular localization.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 10, Lipid-Anchored Membrane Proteins.

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Plasma Membrane Proteins

Which of the following membrane proteins is involved in the 'Chloride-Shift' for CO₂ transport in RBCs?

Physiological transport of carbon dioxide from tissues to lungs uses chloride shift mechanism. CO2 diffusing into erythrocytes hydrated by zinc metalloenzyme carbonic anhydrase II to carbonic acid rapidly dissociating into bicarbonate and proton; proton buffered by deoxygenated hemoglobin promoting Bohr effect increasing oxygen release. Accumulating bicarbonate exported down gradient via anion exchanger Band 3 also termed AE1 SLC4A1 comprising fourteen transmembrane helices functioning as electroneutral one to one Cl- HCO3- antiporter exchanging extracellular chloride inward for intracellular bicarbonate outward known as chloride shift discovered by Hamburger. Exchange rate about ten thousand turnovers per second supports high capacity. At pulmonary capillaries carbonic anhydrase reverse direction reconverts bicarbonate to CO2 for exhalation chloride exits simultaneously restoring ionic balance. Band 3 C-terminal membrane domain also anchors cytoskeleton via ankyrin maintaining shape. Glycophorin sialoglycoprotein and spectrin cytoskeletal lack transport activity. Mutations cause hereditary spherocytosis and distal renal tubular acidosis demonstrating vital transport role. Quantitative transport assays demonstrate high turnover numbers supporting massive CO2 flux required for tissue metabolism and pH regulation.

Ref: Alberts et al., Molecular Biology of the Cell, 6th ed., Chapter 11, Anion Exchanger Band 3 and CO2 Transport.

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Plasma Membrane Proteins

WB2:H25hich membrane protein type is only temporarily associated with the membrane and can be removed using high salt or

Operational definition of peripheral membrane proteins rests on extraction behavior under conditions preserving bilayer integrity. Integral proteins contain hydrophobic stretches of about twenty residues embedding deeply into acyl core requiring non-ionic detergent for solubilization. Peripheral proteins attach superficially through electrostatic attraction between clusters of lysine and arginine and acidic phospholipids phosphatidylserine phosphatidylinositol phosphate, via calcium bridging mediated by C2 domains, or via specific protein-protein interaction with cytoplasmic tails of integral proteins without penetrating hydrophobic interior. Examples include cytochrome c bound to cardiolipin on mitochondrial intermembrane space surface, spectrin ankyrin complex linked to Band 3 cytoplasmic domain, phospholipase C delta PH domain recruited to PIP2, protein kinase C. Treating sealed right-side-out vesicles with high ionic strength one molar sodium chloride screens electrostatic attraction, one hundred millimolar sodium carbonate pH eleven converts vesicles to open sheets releasing trapped proteins, and EDTA chelates divalent calcium disrupting calcium-dependent binding. After ultracentrifugation, released peripheral pool appears supernatant while integral proteins remain pellet-associated.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 10, Peripheral Membrane Proteins and Extraction.

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Lipid Link Protein

What type of lipid linkage does CD59 have?

CD59 protectin MIRL MAC-inhibitory protein exemplifies complement regulation through GPI anchoring. Gene located chromosome eleven encodes one hundred twenty eight amino acids including twenty six residue C-terminal GPI signal peptide removed in endoplasmic reticulum. Preassembled GPI precursor synthesized via stepwise addition of N-acetylglucosamine to phosphatidylinositol, deacetylation, addition of three mannose residues via PIG enzymes, and phosphoethanolamine addition is transferred en bloc by GPI transamidase containing PIG-K catalytic subunit forming amide bond between protein and phosphoethanolamine linked to terminal mannose, core glycan containing glucosamine connecting to inositol phospholipid diacylglycerol embedded in outer leaflet. Resulting anchor confers partition into lipid rafts and rapid lateral diffusion to sites of complement activation where CD59 binds C8 alpha and C9 first transmembrane region blocking polymerization of membrane attack complex pore. Bacterial PI-PLC cleavage releases CD59. Paroxysmal nocturnal hemoglobinuria caused by somatic PIGA mutation disrupting GPI synthesis eliminates CD59 and CD55 causing hemolysis detected by flow cytometry FLAER staining.

Ref: Alberts et al., Molecular Biology of the Cell, 6th ed., Section: GPI-Anchored Proteins – CD59 and Complement Regulation.

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Membrane Transportation

The E1 conformation of Na+/K+ ATPase has:

Post-Albers scheme for P-type ATPases defines two principal conformers interconverting via phosphorylation. E1 state is outward closed, inward open, with N domain positioned to accept ATP and transmembrane domain exposing high affinity sodium sites to cytosol. Detailed structures from shark rectal gland enzyme show three sodium ions coordinated by oxygen atoms in pocket formed by M4, M5, M6 and M8 with micromolar affinity at cytosolic concentration about 10 millimolar, while potassium affinity submillimolar low because pocket incompatible with potassium dehydration geometry. Phosphorylation produces E1P occluded trapping ions, then isomerizes to E2P outward open releasing sodium due to affinity drop thousand fold. In E2, potassium binding sites with different carbonyl arrangement display high potassium affinity from extracellular side where potassium about 4 millimolar, sodium affinity negligible. Dephosphorylation triggers occlusion and transition back to E1. This reciprocal affinity switch ensures ordered antiport. Thus characteristic of E1 conformation is high affinity for sodium and low affinity for potassium as opposed to E2.

Ref: Albers and Post Model, P-Type Pump Cycle, E1 High Na+ Affinity and E2 High K+ Affinity States.

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Protein Sorting and Trafficking

The topology of membrane proteins is determined by:

Topological orientation of membrane proteins, defined by which loops face cytosolic versus exoplasmic or lumenal space, is encoded within polypeptide itself as a set of topogenic signals rather than imposed solely by external receptors. As nascent chain enters Sec61 translocon, hydrophobic segments can function either as start-transfer signal-anchor that opens lateral gate and initiates translocation of downstream region, or as stop-transfer sequence that halts translocation and partitions laterally into lipid bilayer through same gate formed by helices 2b and 7. Positive-inside rule, first documented in bacteria and conserved in eukaryotes, shows distribution of positively charged lysine and arginine residues flanking hydrophobic core strongly influences orientation because moving positive charge across membrane costs more energy and because Sec61 retains basic flanks cytosolically via acidic residues lining pore and anionic phospholipid attraction. Order of successive signal-anchor and stop-transfer elements determines multi-spanning topology polytopic proteins. SRP receptor presence or ATP hydrolysis modulates efficiency of targeting but does not rewrite inherent topological information encoded by charge bias and hydrophobicity profile of signal-anchor and stop-transfer sequences.

Ref: von Heijne G, Annu Rev Biophys 36: 2007, Membrane Protein Topology Determinants.

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