1-27-2010 Cinderella Aquino. Passive vs Active Transport.

CELL BIOLOGY REVIEW

1-27-2010Cinderella Aquino

Passive vs Active Transport

Effect of size, polarity, and charge in diffusion

Figure 11-4a Molecular Biology of the Cell (© Garland Science 2008)

Simple diffusion is limited to small, nonpolar molecules

Diffusion is always movement toward equilibrium (minimum free energy)

Solute size (cut-off size is approx. 200 amu) Generally, membranes are more permeable to

smaller molecules than larger ones For example: Glucose is too large

Solute polarity Permeable to nonpolar, more impermeable to polar

Ion Permeability Membranes are impermeable to ions

Small nonpolar molecules: oxygen, carbon dioxide and ethanol can easily diffuse across membrane

Osmosis Water diffuses to an area of low solute to an area of

high solute

Osmolarity: solute concentration on one side of a membrane relative to that on the other side of the membrane; drives the osmotic movement of water across the membrane

Hypertonic: a solution with a higher solute concentration than inside the cell

Isotonic: a solution with an equal solute concentration than inside the cell

Hypotonic: a solution with a lower solute concentration than inside the cell

Ouabain: inhibitor of sodium/potassium pump

Exposure causes cell swelling

Figure 11-16 Molecular Biology of the Cell (© Garland Science 2008)

Facilitated Diffusion

Carrier proteins (transporters or permeases) bind one or more solute molecules on one side of the membrane and undergo conformational change to deliver solute to the other side of membrane

Channel proteins: form hydrophilic channels, often transport ions Ion channels Porins Aquaporins

Carrier Protein/Transporters/Permeases

Carrier proteins transport either one or two solutes Uniport: single solute Cotransport: two solutes (couple)

Symport: both in the same direction Antiport: solutes are transported in opposite

directions

Uniport

Ex: Glucose Transporter Family or GLUT Permeases

Low cellular glucose is maintained by hexokinase which phosphorylates glucose to glucose-6-phosphate (traps glucose in cell this way, cell lacks transporter for phosphorylated sugars)

Antiporter

Ex: Anion exchange protein/Band 3/ Chloride-bicarbonate exchanger

Solute binding site of the anion exchange protein interacts with different ions on opposite sides of the membrane Necessary to prevent net charge imbalance (one negative

ion in for one negative ion out)

The anion exchanger transport Cl- across the membrane by countertransport with HCO3

- (called the chloride shift)

Important in the transport of carbon dioxide in the body and for helping to regulate pH

Symporter

Ex: Sodium-glucose symporter

Na+-glucose cotransporter: a symporter carrier protein most commonly found in surface absorptive cells of the small intestine mucosa and the simple columnar epithelium of the proximal convoluted tubule of the kidney that transport Na+ across the luminal membrane into the cytoplasm by cotransport with glucose

Cystinuria

Patients cannot transport certain amino acids (including cystine, the disulfide-linked dimer of cysteine) from either urine or the intestine into the blood The resting accumulation of cystine in the urine

in the urine leads to the formation of cystine stones in the kidneys

These crystals and stones can create blockages in the urinary tract and reduce the ability of the kidneys to eliminate waste through urine.

The stones also provide sites where bacteria may cause infections.

Transporter Mutation in Cystinuria

The b0,+ transport system is composed of two separate proteins with specialized functions. One protein, named b0,+AT (for amino acid transporter of neutral and dibasic

amino acids), is responsible for physically moving cystine and the dibasic amino acids from areas where they collect into adjacent tissue.

The other, named rBAT (for related to basic amino acid transporter) is responsible for trafficking the b0,+AT protein to a location in the tissue where it is functional.

Together, they form a complete and functional system.

Without a properly functioning b0,+AT subunit, no cystine transport can occur, and without the rBAT subunit, b0,+AT is unable to get to where it needs to be in order to function properly.

Thus, a defect in either subunit can result in reduced or absent transport of cystine, and thus cause cystinuria.

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Channel proteins facilitate diffusion by forming hydrophilic transmembrane channels

Three kinds of channel proteins: Ion channels: Transmembrane proteins that allow rapid passage of

specific ions

Voltage-gated Ligand-gated Mechanosensitive

Porins: Transmembrane proteins that allow rapid passage of various solutes Beta barrel transmembrane region creates water-filled pore at its center

Aquaporins: Transmembrane channels that allow rapid passage of water Can facilitate transport at a rate of several billion water molecules per

second Found in certain tissues such as the proximal tubules of the kidneys that

reabsorb water as part of urine formation

Four types of Transport ATPases

P-type ATPases (P for phosphorylation): Reversibly phosphorylated by ATP as part of the transport mechanism. They are responsible for maintaining an ion gradient across the membrane Example: Na/K pump and proton pump in stomach

F-type ATPases (F=factor): ATP synthases. Proton transporter found in bacteria, chloroplasts and in mitochondria.. Not only can ATP be used as an energy source to generate and maintain electrochemical gradients, but such gradients can be used as an energy source to synthesize ATP

V-type ATPases (V=vesicle): Pump protons into such organelles as vesicles, vacuoles, lysosomes, endosomes, and the Golgi complex in a phosphorylation independent manner Structurally related to F-type ATPases

ABC-type ATPases (ATP-binding cassette): Large superfamily. Handles a wide variety of solutes. (ions, sugars, AA, peptides and polysaccharides) Example: Multidrug resistance transport protein found in tumor cells, may

help these cells evade drug therapy. CFTR chloride ion channel involved with cystic fibrosis

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ATPases

ABC Transporter Superfamily Each family member contains two highly conserved ATP-binding cassettes

Multidrug resistance proteins MDR1: a uniporter carrier protein found in hepatocytes of the liver that transports cholesterol into the

bile canaliculus MDR2: a uniporter carrier protein found in hepatocytes of the liver that transports phospholipids into

bile canaliculus MDR1 and MDR2 are expressed by human cancer cells and unfortunately confers

resistance to cancer chemotherapeutic drugs by transporting the hydrophobic drugs out of the cancer cell

Chloroquine transporter is expressed by Plasmodium falciparum (which causes malaria) and confers resistance to the antimalarial drug chloroquine by transporting the drug out of P. falciparum.

Cystic fibrosis transporter: The CFTR gene encodes for a chloride transporter. Mutation in this gene causes cystic fibrosis

Flippase removes phosphatidylethanolamine and phosphatidylserine from the outer leaflet of the cell membrane and uses the energy from ATP hydrolysis to flip them into the inner leaflet.

Multispecific organ anion transporter: a uniport carrier protein found in hepatocytes of the liver that transports bilirubin glucuronide (bile pigment) and glutathione into the bile canaliculus.

Biliary acid transporter: a uniport carrier protein found in hepatocytes of the liver that transports bile salts into the bile canaliculus 20

CYSTIC FIBROSIS Caused by an autosomal recessive mutation in the

cystic fibrosis transmembrane conductance regulator (CFTR) gene An ABC transporter family membre Most common CFTR mutation in Caucasians is DF508

Mechanism: The mutation results in inability of cells to

transport chloride and water to body secretions Results in increased frequency of respiratory

infections Also causes pancreatic insufficiency Males often are sterile End stage, progressive lung disease is the

principle cause of death

ATP Synthase (F-type pump)

Makes ATP from ADP + Pi, utilizing the proton gradient across the inner membrane of the mitochondria

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Active transport: Protein-mediated movement up the gradient

Active transport requires expenditure of energy! Unlike simple and facilitated diffusion (both

nondirectional), active transport has directionality usually a unidirectional process

Three major functions of Active Transport:1) Uptake of essential nutrients2) Removal of secretory products and waste3) Maintain nonequilibrium intracellular concentrations of

ions The coupling of active transport to an energy source may

be direct or indirect Direct active transport depends on four types of

transport ATPases Indirect active transport is driven by ion gradients

2/3 of body’s energy consumed to maintain gradients of ions such as H+, K+, Na+ and Ca+

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Direct versus Indirect

Direct active transport: the accumulation of solute molecules or ions on one side of the membrane is coupled directly to an exergonic chemical reaction (ATP hydrolysis)

Indirect active transport: depends on the cotransport of two solutes, with the movement of one solute down its gradient driving the movement of the other solute up its gradient (usually sodium or proton ions) Animal cells usually depend on Sodium ion (Na+)

gradients as the driving force for indirect active transport

Plant, bacteria and fungi usually depend on proton (H+) gradients

Direct Active Transport: Na/K Pump

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Na+-K+ ATPase: an antiporter carrier protein found in almost all cells that pumps Na+ out of cell and K+ into the cell to maintain a low intracellular [Na+].

P-type pumpOubain is a specific inhibitor that competes for K+-binding site. Cardiac glycosides (digoxin and digitoxin) are Na+-K+ ATPase blockers that elevate intracellular Na+ levels within cardiac myocytes. The elevated Na+ overwhelms the Na+- Ca2+ Exchanger so that more Ca2+ can be reaccumulated by the sarcoplasmic reticulum. During the next contraction, more Ca2+ is released from the sarcoplasmic reticulum, which increased the force of contraction. Cardiac glycosides are used in congestive heart failure to increase the force of contraction

Indirect Transport: Na/glucose symporter

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Na+-glucose cotransporter: a symporter carrier protein most commonly found in surface absorptive cells of the small intestine mucosa and the simple columnar epithelium of the proximal convoluted tubule of the kidney that transport Na+ across the luminal membrane into the cytoplasm by cotransport with glucose.


Transcellular Transport of Glucose

The transcellular transport of glucose across an intestinal epithelial cell depends on the nonuniform distribution of transporters in the cell’s plasma membrane. The process shown here results in the transport of glucose from the intestinal lumen to the extracellular fluid.

Glucose is pumped into the cell through the apical domain of the membrane by a Na+ - powered glucose symporter.

Glucose passes out of the cell (down its concentration gradient) by passive movement through a different glucose transporter in the basal and lateral membrane domains.

The sodium gradient driving the glucose symport is maintained by the sodium pump in the basal and lateral plasma membrane domains, which keeps the internal concentration of Na+ low.

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Transport of Glucose Into Cells

Glucose cannot diffuse directly into cells, but enters by one of two transport mechanisms:

1. an Na+-independent, facilitated diffusion transport system Or

2. an Na+-monosaccharide co-transporter system.

Cells and Transport processes

Membrane potential: voltage across a membrane created by ion gradients; usually, the inside of a cell is negatively charged with respect to the outside

Electrochemical gradient: transmembrane gradient of an ionic species, with both an electrical component due to charge separation and a concentration component

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***As a result of so much selective pumping of ions in and out ofthe cell, the cytoplasm inside the cell has a very different ionic

composition than the extracellular fluid

***The resulting membranepotential inside the cell isnegative

Resting membrane potential (Vm) Electrochemical equilibrium: condition in which a transmembrane concentration gradient of a specific ion is balanced with an electrical potential across the same membrane, such that there is no net movement of the ion across the membrane

The membrane potential in animal cells depends mainly on K+ leak channels and the K+ gradient across the plasma membrane The attraction of the negatively charge molecules helps keep K+ in the cell, yet

the concentration gradient encourages it to leave the cell. The resting membrane potential is a consequence of these two influences

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Transport of large molecules:

Endocytosis – Uptake of macromolecules from the extracelluar surroundings by localized regions of plasma membrane Phagocytosis – endocytosis of large particulate

substances Pinocytosis – endocytosis of fluid and dissolved

solutes Receptor-mediated – binding of ligands to receptors

triggers vesicle formation Exocytosis – secretion of macromolecules by transport

vesicles Endocytosis & exocytosis are mechanisms that involve

movement into and out of the lumen of the endomembrane system Not movement directly across membrane That is, substances enter the Endomembrane System but not the

Cytoplasm 32

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INTRACELLULAR COMPARTMENTS


Proteins can move between compartments in different ways

Almost all protein synthesis begins on ribosomes in the cytosol

Protein’s fate depend on their amino acid sequence, which can contain sorting signals

35Table 12-3 Molecular Biology of the Cell (© Garland Science 2008)

Protein traffic in the cell1. Gated transport

Movement of proteins from cytosol into the nucleus via nuclear pores

2. Transmembrane transport

Protein translocators directly transport specific proteins across a membrane from the cytosol into a space that is topologically distinct

3. Vesicular transport

Membrane-enclosed transport intermediates ferry proteins from one compartment to another

36Figure 12-6 Molecular Biology of the Cell (© Garland Science 2008)


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Anterograde and Retrograde transport

Anterograde transport: movement of material from the ER through the Golgi complex toward the plasma membrane

Retrograde transport: movement of vesicles from the Golgi cisternae back toward the ER

A “road-map” of the biosynthetic-secretory and endocytic pathways

Key: Endocytic pathway is in green Biosynthetic pathway is in red Retrieval pathways are in blue (Retrograde)

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Figure 13-3 Molecular Biology of the Cell (© G-secretoryarland Science 2008)

There are various types of coated vesicles Different coat proteins select

different cargo and shape the transport vesicles that mediate the various steps in the biosynthetic-secretory and endocytic pathways

Three examples (MUST KNOW!) Clathrin-coated vesicles mediate

transport from the Golgi and from the plasma membrane

COP I bud from the Golgi complex

COP II bud from the ER



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Clathrin-coated vesicles

Protein coats may help with forming vesicles from flat membranes, targeting and interactions with cytoskeleton

Clathrin-coated vesicles are surrounded by lattices composed of clathrin and adaptor protein Components of clathrin lattices: triskelion (heavy and light

chains) Adaptor protein (assembly protein) bind transmembrane

receptors, confer specificity in budding and targeting, mediate attachment of clathrin. AP are sites of regulation

The assembly of clathrin coats drive the formation of vesicles from the plasma membrane and TGN

The accumulation of clathrin forming hexagonal and pentagonal lattice, curves budding vesicle.

Dynamin: a cytosolic GTPase required for coated pit constriction and closing of the budding vesicle

An uncoating ATPase is involved with uncoating vesicles after they bud from membrane

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COP I- and COP II- coated vesicles connect the ER and Golgi complex cisternae

COP I coated vesicles COP I are composed of COPI and ADP ribosylation

factor (ARF) “Fuzzy” coats. Not lattices COP I facilitates retrograde transport of proteins

from the Golgi back to the ER, as well as between cisternae of the Golgi. They DO NOT bud from the ER

Assembly of COPI coat is mediated ARF (GTP binding protein)

ARF interact with guanine nucleotide exchange factor, switches GDP for GTP, inserts hydrophobic tail into membrane, bind COPI multimers, assemble coat

COP II coated vesicles Transport of material from the ER to the Golgi

Phosphoinositides mark organelles and membrane domains(A, B) The structure of PI shows the

free hydroxyl groups in the inositol sugar that can in principle be modified

(C) Phosphorylation of one, two or three of the hydroxyl groups on PI by PI and PIP kinases produce a variety of PIP species. They are named according to the ring positions (in parentheses) and the number of phosphate groups (subscript) added to PI. PI (3,4)P2 is shown

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(D) Animal cells have several PI and PIP kinases and a similar # of phosphatases, which are localized to different organelles, where they are regulated to catalyze the production of particular PIPs

(E,F) Phosphoinositides head groups are recognized by protein domains that discriminate between the different forms. In this way, selected groups of proteins containing such domains are recruited to regions of membrane in which these phosphoinositides are present

The intracellular location of phosphoinositides

Different types of PIPs are located in different membranes and membrane domains, where they are often associated with specific vesicular transport events For example, the membrane of

secretory vesicles contains PI(4)P. When the vesicles fuses with the

plasma membrane, a PI 5-kinase that is localized there converts the PI(4)P into PI(4,5)P2.

The PI(4,5)P2, in turns, helps recruit adaptor proteins, which initiate the formation of clathrin-coated pit, as the first step in clathrin-mediated endocytosis.

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Monomeric GTPase: Control Coat Assemby

GTPases are inactive when bound to GDP GTPases are ACTIVE when bound to GTP Guanine-nucleotide-exchange factors

(GEFs) activates monomeric GTPases by having the GTPases release GDP and then bind GTP

GTPase-activating proteins (GAPs) inactivate the monomeric GTPases by triggering the hydrolysis of bound GTP to GDP

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Rab Proteins

Monomeric GTPases Play role in specificity of vesicular

transport to ensure they interact with the correct target membranes

Vesicle fated for different destinations have distinct members of the Rab family associated with them.

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The SNARE hypothesis connects coated vesicles and target membranes

Vesicles must be properly targeted The SNARE hypothesis: model explaining how membrane

vesicles fuse with the proper target membrane; based on specific interactions between v-SNAREs and t-SNAREs

Specificity of process comes from vesicle-SNAREs, target-SNAREs, Rab GTPases and tethering proteins

NSF (N-ethylmaleimide sensitive factor) uncouples SNAREs after fusion and SNAPs (soluble NSF attachment receptor) mediate fusion

Best-studied SNAREs: those that mediate docking of synaptic vesicles with the presynaptic membrane - targets of bacterial neurotoxins responsible for botulism and tetanus.

Endomembrane System Endoplasmic reticulum The Golgi complex Endosomes Lysosomes The nuclear envelope and perinuclear space Transport vesicles


The Rough ER

membrane

Boundribosomes

*note thatribosomesare onoutside of

Pancreatic acinar cell ER

Rough ER is involved in the biosynthesis and processing of proteins

Rough ER is the site of protein synthesis for membrane-bound and soluble proteins of the endomembrane system as well as for proteins on the cell membrane and those that are secreted

Most proteins enter endomembrane system are inserted into the rough ER lumen cotranslationally A Signal-Recognition Particle (SRP) directs ER signal

sequences to a specific receptor on the rough ER membrane

ER is the site for the initial steps of addition of carbohydrate groups to glycoproteins, the folding of polypetides, and the assembly of multimeric proteins

Rough ER is the site of hydroxylation of proline and lysine during collagen synthesis

Protein Quality Control- abnormal proteins are targeted for degradation ER-associated degradation (ERAD): abnormal proteins are

exported from the ER for degradation by cytosolic proteasomes

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Combination of Start-transfer and Stop-transfer signals determine the topology of multipass transmembrane proteins

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A

D

C

B

Most proteins synthesized in the rough ER are glycosylated by the addition of a common N-linked oligosaccharide

A precursor oligosaccharide complex is transferred en bloc to proteins in the ER

The precursor oligosaccharide core complex is constructed on a special lipid molecule (dolichol) The core complex is composed of

14 sugars: 2 GlcNACs, 9-mannoses and 3 glucoses

90% of all glycoproteins are N-linked glycosylated

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Steps Involved with N-linked Glycosylation

Synthesis of core oligosaccharide complex

Transfer of Oligosaccharide core from dolichol to target protein

53Figure 12-51 Molecular Biology of the Cell (© Garland Science 2008)Figure 12-52 Molecular Biology of the Cell (© Garland

Science 2008)

Oligosaccharides are used as tags to mark the state of protein folding

The pattern of N-linked glycosylation is used to indicate the extent of protein folding, so that proteins leave the ER only when they are properly folded.

ER chaperones calnexin and calreticulin recognize core oligosaccharide structures missing two of the three glucose molecules

After the third glucose is removed from the oligosaccharide structure, calnexin and calreticulin can no longer bind and the properly folded protein can leave the ER

Proteins that do not fold or oligomerize correctly are translocated back into the cytosol, where they are deglycosylated, ubiquitylated, and degraded in proteosomes

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Composition of core oligosaccharide structure influences protein’s fate in ER

The ER chaperone proteins bind to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the ER

Removal of the terminal glucose by glucosidase releases the protein from calnexin

A glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: If the protein is still incompletely folded, the enzyme

transfers a new glucose to the core structure This allows calnexin or calreticulin (a soluble protein)

to bind and help refold the misfolded protein

Misfolded proteins are translocated to the cytosol, ubiquinated and destroyed by proteosomes

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Some proteins acquire an attached glycosyl-phosphatidylinositol (GPI) anchor in the ER

Immediately after the completion of protein synthesis, the precursor protein remains anchored in the ER membrane by a hydrophobic C-terminal sequence

An enzyme in the ER cuts the protein free from its membrane-bound C-terminus and simultaneously attaches the new C-terminus to an amino group on the preassembled GPI intermediate


Some membrane proteins lose their transmembranedomain and gain a GPI anchor

A GPI anchor can act as a sorting signal to direct these membraneproteins to special regions of the plasma membrane (caveolae)

Exit from the ER is controlled to ensure protein quality Proteins with ER retention signal stay in or are returned to ER Secreted proteins are packaged into vesicles and sent to the

Golgi complex after they are properly folded Incorrectly folded proteins are retained in the ER by

chaperone proteins until they are properly folded or destroyed Most common mutation of CFTR (DF508) that causes cystic

fibrosis cannot be properly folded in the ER and is destroyed. The mutated CFTR would be functional if it could make it to the cell surface –

but never does!

58Figure 15-24 Essential Cell Biology (© Garland Science 2010)

The size of the ER is controlled by the amount of protein that flows through it

Excessive accumulation of misfolded proteins in the ER will activate the unfolded protein response (UPR)

The UPR program prompts the cell to produce more ER, including molecular machinery to restore proper protein folding and processing

If the cell is too overwhelmed, the UPR program can induce apoptosis In some cases of type II diabetes,

pancreatic cells that secrete insulin undergo apoptosis because their ER reached maximum capacity for synthesizing and processing insulin . Thus the diabetes becomes worse

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Figure 15-25 Essential Cell Biology (© Garland Science 2010)

1-27-2010 Cinderella Aquino. Passive vs Active Transport.

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