Protein Traffic and Signal Recognition in Cellular Compartments, Summaries of Biology

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G. J. Tortora, B. H. Derrickson,

Biology and physiology

Table of Contents Lecture 15 Intracellular Transport ................................................................................................................. 2 Covered in presentation but not in lecture ............................................................................................... 3 Lecture 16 ...................................................................................................................................................... 4 Lecture 17 ...................................................................................................................................................... 7 Lecture 18 .................................................................................................................................................... 13 Key: Text Topics/details covered in lecture but not seemingly relevant to exam material, thus skipped Text Topics/details not necessary to know for exam

▪ A cytosolic protein , RAN-GAP (RAN GTP-ase activating protein) activates hydrolysis of GTP attached to RAN. ▪ This creates a gradient of RAN-GTP across the nuclear pore – with more RAN GTP inside the nucleus than outside. ▪ RAN-GTP binds to nuclear import receptors after they diffuse through the nuclear pore and into the nucleus ▪ causes them to release their cargo proteins, which therefore accumulate inside the nucleus. ▪ RAN-GTP has the opposite effect on nuclear export receptors , causing them to bind their cargo. ▪ They then diffuse through the pore into the cytosol. ▪ Import depends on the receptor NOT binding to RAN when it binds to its cargo. When the receptor gets into the nucleus it binds to the RAN and release cargo ▪ Export relies on both receptor and RAN bound at the same time in the nucleus, both are released when receptor gets into cytosol ▪ Focus on nuclear import o Nuclear transport as a means of gene regulation ▪ The activity of some gene regulatory proteins is controlled by keeping them out of the nuclear compartment until they are needed there. ▪ In many cases, this control depends on the regulation of nuclear localization and export signals ; these can be turned on or off, often by phosphorylation of adjacent amino acids. o Ex. NFAT and Nuclear Transport ▪ T cell activated via antigen binding ▪ Ca2+^ levels increase due to Ca2+^ channel opening ▪ This rise in Ca2+^ levels activates the protein phosphatase, calcineurin ▪ Calcineurin dephosphorylates NF-AT ▪ Dephosphorylation of NFAT causes a conformational change which exposes a nuclear import sequence on the protein’s surface. ▪ NFAT enters the nucleus, where it triggers gene expression appropriate to the T- cells role in the immune response. Covered in presentation but not in lecture

• Movement of proteins between cellular compartments: Transmembrane transport o protein traffic between the cytosol and an organelle that is topologically different ▪ Cytosol → ER ▪ Cytosol → mitochondria - Neither the mitochondrial nor chloroplast genomes contain information necessary to code for all of their proteins. - They rely on the import of their proteins from the cytosol following synthesis. o occurs through membrane-bound protein translocators ▪ The transported protein molecule usually must unfold to snake through the translocator
• Movement of proteins between cellular compartments: vesicular transport

o protein traffic among topologically equivalent organelles ▪ ER ↔ Golgi ▪ Golgi ↔ Endosomes ▪ Endosomes ↔ Lysosomes ▪ Endosomes ↔ Plasma Membrane o occurs through membrane-enclosed transport intermediates called VESICLES

• …
• Newly synthesized (precursor) Mitochondrial Proteins in the cytosol are surrounded by protein- folding chaperones that prevent them from aggregating. o Most common chaperone: Hsp
• Mitochondrial versions of these chaperones also exist and help these precursor proteins fold into 3D structures once they enter the mitochondria. Lecture 16 10/25/
• Mitochondrial transport o Mitochondrial proteins are first fully synthesized as precursor proteins in the cytosol and then translocated into mitochondria o Most of the mitochondrial precursor proteins have a signal sequence at their N terminus that, when folded forms an amphipathic alpha helix o Charged residues cluster on one side o Uncharged residues cluster on the other side o Protein translation across mitochondrial membranes is mediated by multi-subunit complexes that function as protein translocators: ▪ The TOM complex functions across the outer membrane ▪ Two TIM complexes, the TIM23 and TIM22 complexes, function across the inner membrane
• Mitochondrial protein translocators o ALL nucleus-encoded mitochondrial proteins must first enter via TOM o Helps insert transmembrane proteins into outer mitochondrial membrane o Transmembrane proteins with a β-barrel structure are transferred to the SAM complex for proper folding. o TIM 23 spans both outer and inner mitochondrial membranes o Transports: ▪ soluble proteins into MATRIX ▪ membrane proteins into inner mitochondrial membrane o Import ATPase complex binds to and pulls proteins through TIM23 channel
• Proteins entering the ER undergo CO-translational translocation o imported into the ER as they are being synthesized ▪ no requirement for additional ATP to pull the protein into the ER ▪ as you extend the protein, the force generated by extending the protein pushes it across the transporter into the ER o What types of proteins require co-translational translocation? ▪ Water soluble (non membranous) proteins destined to:

• Coat Proteins Represent the Initial Step in Vesicle Formation o Name a protein coat complex – Clathrin, COPI, COPII o Transport vesicles bud off as coated vesicles that have a distinctive cage of proteins covering their cytosolic surface. o Before the vesicle fuses with a target membrane, the coat is discarded , to allow the two cytosolic membrane surfaces to interact directly and fuse. o Different coat proteins are involved in transport between different organelles: ▪ COPII coats ER-to-Golgi vesicles ▪ COPI coats vesicles moving from Golgi to ER, Golgi to plasma membrane (secretory vesicles), and within the Golgi ▪ Clathrin is mainly involved in transport to/from and within the endosomal compartments
• Phospholipids containing inositol head groups mark organelles and membrane domains o Inositols can get phosphorylated at various locations by different lipid kinases (often located in distinct organelles, providing specificity)
• (Phosphatidyl inositol) PI’s can recruit various proteins that possess lipid binding domains o These lipid binding domains usually only recognize a specific type of PI
• Regulation of coat assembly and vesicle stability o Adapter proteins bind to membrane proteins and recruit coat proteins ▪ Often bind to cargo receptors – transmembrane proteins that bind soluble cargo proteins for transport o Coat recruitment GTPases control coat assembly ▪ Monomeric GTPases regulate many steps in vesicular traffic ▪ Sar- 1 regulates COPII assembly ▪ Arf proteins regulate COPI and clathrin assembly o Active Sar1-GTP then promotes the assembly of coat complexes. o GTP hydrolysis causes coat disassembly after budding. ▪ Sar1-GDP = cytosol = inactive ▪ Sar1-GTP = ER membrane-bound = active
• Vesicle docking and targeting o Membrane traffic needs to proceed in an orderly way ▪ Transport vesicles must be highly selective in recognizing the correct target membrane with which to fuse. o Specificity in targeting: surface markers that identify vesicles according to their origin and type of cargo. o Target membranes display complementary receptors that recognize the appropriate markers.
• Recognition of donor vesicles by acceptor membranes is controlled mainly by two classes of proteins: SNAREs and Rabs. o SNARE proteins ▪ provide specificity ▪ catalyze the vesicular fusion with the target membrane. ▪ v-SNAREs: vesicle ▪ t-SNAREs: target membrane

o Rabs (GTPases): ▪ Work together with other proteins to regulate the initial docking and tethering of the vesicle to the target membrane ▪ See Table 13- 1 for list of some Rabs

• Entry into vesicles leaving the ER is usually a selective process o Cargo Recruitment ▪ Membrane proteins have exit signals in their cytosolic ‘tails’ that are recognized by coat proteins ▪ Soluble Proteins bind to cargo receptors that have exit signals in their cytosolic ‘tails’
• Transport from the ER to the Golgi Apparatus Is Mediated by Vesicular Tubular Clusters o Transport vesicles leaving the ER fuse together to form intermediate compartments called vesicular tubular clusters o These clusters ▪ travel towards the cis Golgi via motor proteins on microtubule tracks ▪ generate coated vesicles going back to the ER (COPI coat) – retrograde transport
• ER retrieval signals: o Membrane ER resident proteins retrieval signals in their cytosolic tails ▪ recognized by COPI coat proteins o Soluble ER resident proteins ▪ Retrieval signals within their structure ▪ Bind to receptors ▪ EX: KDEL sequences bind to KDEL receptors
• Two current models for how cargo travels through the Golgi: o CISTERNAE = STATIC ▪ Vesicles travel between them o CISTERNAE = DYNAMIC ▪ Move upward, changing their properties slightly as they migrate ▪ Vesicular tubular cluster becomes new cis face of golgi, old cis face becomes medial cisternae, old trans face becomes trans golgi network and can move away/bud off vesicles and turn over to become a new trans face Lecture 17 10/30/
• Transport from the Trans Golgi Network to the Cell Exterior: Exocytosis o Many proteins leaving the Golgi need to get to the plasma membrane. o Vesicles carrying such proteins fuse with the plasma membrane via exocytosis. o Vesicle cargo ▪ membrane proteins and the lipids become part of the plasma membrane ▪ soluble proteins are secreted into the extracellular space.
• Transport from the Trans Golgi Network to the Cell Exterior: Two Pathways o Constitutive secretory pathway ▪ Never-ending, occurring all the time o Regulated secretory pathway ▪ Proteins are sorted in the trans golgi network and retained for future use

• The budding and internalization of vesicles from the plasma membrane is called endocytosis. o Vesicle cargo ▪ membrane proteins and the lipids are removed from the plasma membrane - some will be Recycled back to the surface - some will be Degraded ▪ soluble proteins - from extracellular space - carried in the lumen ▪ common membrane protein cargo: receptors (and their ligands) ▪ budding in from the plasma membrane is topologically equivalent to budding out from the ER/golgi
• Types of Endocytosis o Distinguished on the basis of the size of the endocytic vesicles formed. ▪ Phagocytosis (“cellular eating”) - the ingestion of large particles , such as microorganisms or dead cells via vesicles called phagosomes (generally >250 nm in diameter). ▪ Pinocytosis (“cellular drinking”) - the ingestion of fluid and solutes - Vesicles called pinocytic vesicles (about 100 nm in diameter). - Includes receptor-mediated endocytosis o Most eukaryotic cells are continually ingesting fluid and solutes by pinocytosis
• Ex. Cholesterol gets into cells via receptor-mediated endocytosis
• Ex. Epidermal growth factor receptor (EGFR) gets degraded, along with its ligand, but transferrin receptor (TfnR) gets recycled to plasma membrane
• Transcytosis: Molecules internalized at one end of a ‘polarized’ cell are transported to a different end.
• Membranes as Barriers o Biological membranes serve as semipermeable barriers o Nonpolar molecules can diffuse freely, but polar/charged solutes are unable to cross without assistance o Transmembrane protein channels and transporters serve to allow polar/charged solutes to cross membranes ▪ Channels allow diffusion down a concentration gradient ▪ Transporters use conformational changes to move substrates across the membrane, and may transport down ( passive transport) or up ( active transport) a concentration gradient
• Diffusion o HIGH concentration → LOW concentration ▪ Going from a more to less ordered state → increase in entropy o DOWN concentration gradient: ▪ concentration gradient– difference in concentration of a solute in one region compared to another o The process or movement of any molecule or ion moving down or up a concentration gradient requires a change in free energy

• Which is true about diffusion? Molecules sometimes move towards other molecules and sometimes away from other molecules o Random motion will result in spreading out of molecules over time
• Diffusion of CHARGED substances across cell membranes o Diffusion is net movement of charged solute down its electrochemical gradient o sum of: ▪ concentration gradient ▪ electrical gradient – electrical potential difference between 2 sides of membrane
• Electrochemical differences are additive
• So, ΔG (Gibbs free energy) can tell us whether a substance will move passively (no energy needed) or actively (energy input required) across a membrane. o If positive, the value of DG^ (net flux INTO cell) is the^ amount of energy required to move^ a mole of solute up its electrochemical gradient and into the cell. o If negative, DG^ (net flux INTO cell) is the maximum^ amount of energy available to drive another process that is coupled to the diffusion of a solute down its electrochemical gradient and into the cell.
• Ways molecules can get across a membrane: simple diffusion o Diffusion across lipid bilayer (independent of proteins) o Limited by: ▪ Polarity ▪ Charge ▪ Size
• Diffusion across membranes: Permeability o In the absence of proteins, the lipid bilayer allows free diffusion of select types substances down their concentration gradients: ▪ hydrophobic molecules ▪ Small polar molecules o Restricts the diffusion of other types of substances: ▪ ions ▪ larger polar molecules o The membrane = semipermeable barrier. o The membrane must be able to maintain concentration differences between the internal and external environments. o Health of the cell requires that material transport be a regulated process.
• Transport Proteins o Cells have evolved membrane proteins whose function is to transport small molecules and ions. o Membrane proteins mediate 2 basic types of movement across cell membranes: ▪ Passive Transport ▪ Active Transport
• Simple Diffusion vs Transporter-mediated diffusion o Simple diffusion:

▪ Temperature

• temperature - sensitive channels
• Passive transport – facilitated diffusion o Transport of neutral, polar molecules larger than water or urea, such as glucose - or charged molecules such as amino acids (rather than simple ions) o not coupled to any external energy source, such as ATP ▪ this is still passive transport even though it requires a shape change in the transporter to get across the membrane) ▪ the direction of net flux follows the electrochemical gradient for whatever molecule is transported. o solute molecule binds tightly to a highly specific site on the protein and causes a conformational change in the transporter protein. ▪ Transition between A and B is random/reversible, does not depend on whether solute binding sites are occupied. o Facilitated transport proteins: ▪ Like enzymes, do not alter DG for transport – movement is always down the electrochemical gradient for the solute - they just speed up movement ▪ Have a relatively slow turnover compared to channels
• maximal transport rate ~1000 molecules per transport protein per second
• compared to 1 million molecules for channels
• Facilitated diffusion – Glucose transport o Humans have 5 related glucose facilitated transport proteins, One of these, GLUT4 is common to insulin-responsive cells o Insulin promotes the insertion of GLUT4 transporters into the membrane of target cells, promoting glucose uptake
• Active transport o Movement of solutes UP their electrochemical gradients. o Can create and maintain concentration gradients of solutes across membranes. o Can sometimes directly contribute to membrane potentials (can be “electrogenic”) if the transported solutes carry a net charge across the membrane.
• Types of active transport o Three main types, depending on the energy source ▪ ATP-ase pumps.
• Couple movement of solutes to ATP hydrolysis ▪ Other pumps that use diverse energy sources (e.g. light, oxidation of NADH) ▪ Coupled transporters
• link the movement of one solute up its electrochemical gradient to the movement of another solute down its electrochemical gradient. Their energy source is therefore an existing electrochemical gradient.
• Coupled transporters o (Uniport: Transport one molecule) o Symport: Transport two molecules in the same direction o Antiport: Transport two molecules in different directions

Lecture 18 11/01/

• Types of ATP-driven pump proteins: P-type Pumps o “P” stands for the use of a high energy phosphoprotein intermediate o Multipass transmembrane proteins o Pump H+, K+, Na+, Ca2+
• P-type Pump Example 1: Ca2+ ATPase o Like Na+, Ca2+^ ions are kept at very low concentrations in the cell relative to the extracellular environment: ▪ Inside the cell: [Ca2+] ~ 0.1μM ▪ Outside the cell: [Ca2+] ~ 103 μM o Even the smallest Ca2+^ cytosolic influx can lead to a massive increase in cell levels. o Regulated Ca2+^ influxes form the basis of a variety of cell signaling events. o Helps maintain a very low resting Ca2+^ concentration in the cytosol. o Transports one Ca2+^ ion out of the cell for every ATP molecule consumed. o Plasma membrane, smooth ER, sarcoplasmic reticulum (muscle ER) o Creates a calcium ion store with in the smooth ER
• P-type Pump Example 2: Na+/K+ Pump o Each cycle of the sodium/potassium pump (Na/K ATP-ase) transports: ▪ 3 Na+ out of a cell ▪ 2 K+ into a cell - Causes a net +1 charge out of the cell per cycle - Electrogenic – producing a change in the electrical potential ▪ All for the hydrolysis of only one ATP molecule o The pump has specific binding sites for sodium and potassium. o The binding and release of sodium and potassium ions on either side of the membrane is coupled to ATP-hydrolysis o Each step in the hydrolysis of ATP and the binding of the transported ions drives conformational changes that create a pumping cycle by alternately exposing the ion binding sites to one side of the membrane and then the other. o Na+ binds o ATP hydrolysis leads to phosphorylation of the pump o Conformational change → release of Na+ o Binds K+ o Causes dephosphorylation of the pump o Conformational change o Unphosphorylated pump has higher affinity for Na+
• Types of ATP-driven pump proteins: V-type Pumps o Made up of multiple subunits o Use ATP, but not via a phosphorylated intermediate o (the phosphate is released, not added to the pump). o V type pumps are found in membranes of: ▪ Lysosomes ▪ Synaptic vesicles

• T-helper cell & B cell
• Signaling molecules: Overall action o #1: Extracellular signals can act slowly or rapidly to alter the function of a target cell o #2: Each cell is programmed to respond to specific combinations of extracellular signal molecules - live or die ▪ Most of the cells in a complex animal are also programmed to depend on a specific combination of signals simply to survive. ▪ When deprived of these signals (in a culture dish, for example), a cell activates a suicide program → apoptosis ▪ Because different types of cells require different combinations of survival signals, each cell type is restricted to different environments in the body. o #2: Each cell is programmed to respond to specific combinations of extracellular signal molecules - divide, differentiate, do ▪ A typical cell in a multicellular organism is exposed to hundreds of different signals in its environment. ▪ These signals can act in many millions of combinations. ▪ A cell may be programmed to respond to one combination of signals by growth/division ▪ to another combination by differentiating ▪ and to yet another by performing some specialized function such as contraction or secretion. o #3: Different cells can respond differently to the same extracellular signal molecule – cells interpret signals ▪ Cellular responses vary according to:
• Unique collection of receptor proteins the cell possesses
• the intracellular signaling machinery by which the cell integrates and interprets what it receives ▪ determine the particular subset of signals to which a cell can respond ▪ The same signal molecule often has different effects on different target cells. ▪ Ex. acetylcholine o #4: The same signaling molecule can have different concentration-dependent effects on the same cell type ▪ So the AMOUNT of signal can dictate what happens within the same cell. ▪ Embryonic Development
• molecules called morphogens diffuse out from signaling centers in developing tissues, creating a morphogen concentration gradient.
• Cells adopt different fates depending on their position in the gradient. In this way layers of cells develop, each with a different function
• Cell communication is like human communication. Paracrine signaling is like talking to people at a cocktail party
• Signaling molecules: types of signals o Cells are specialized to receive and respond to a wide variety of stimuli o Mechanical ▪ adhesion to substrates, membrane distortion, sound

o Light o Heat o Chemical ▪ amino acids, small peptides, and proteins ▪ nucleotides ▪ steroids ▪ fatty acid derivatives ▪ dissolved gases: nitric oxide, carbon monoxide.

• Lipid soluble signals o Many signaling molecules are lipid soluble and can simply diffuse across the plasma membrane. o Example: steroid hormones & gaseous signaling molecules (e.g. nitric oxide) o Steroid hormones vary in chemical structure, but all are synthesized from cholesterol. o Cortisol – adrenal glands ▪ Influences metabolism o Sex hormones – ovaries and testes ▪ Determine secondary sex characteristics that distinguish males and females o Vitamin D – skin in response to sunlight ▪ Regulates Ca2+^ uptake/excretion o Thyroxine (non-steroid, tyrosine derivative)– thyroid glands ▪ Regulates metabolism
• Diffusion across membranes: Permeability o In the absence of proteins, the lipid bilayer allows free diffusion of select types substances down their concentration gradients: ▪ hydrophobic molecules ▪ Small polar molecules o Restricts the diffusion of other types of substances: ▪ ions ▪ larger polar molecules o Lipid soluble signals and their receptors ▪ Steroid hormones: - travel to their target cells via carrier proteins - bind to intracellular receptors, which can either be cytosolic or nuclear
• Intracellular receptors that bind hormones o The receptors for steroid hormones are members of the nuclear receptor subfamily o Prior to ligand binding, these receptors are bound to an inhibitory protein and are inactive o Ligand binding causes a conformational change in the receptor, which causes the inhibitory protein to dissociate. o Exposes a site which binds to the promoter region upstream of a specific targeted gene. o Transcription of that gene is then increased, producing specific proteins that cause changes in cell behavior o Different cells respond differently to hormones because: ▪ Only certain cells have nuclear receptors for each hormone

o Enzymes activated by a receptor protein often form protein complexes called ‘signaling complexes’, which regulate Speed/Efficiency/specificity of a cellular response o These complexes can be organized around a “scaffold” protein o These complexes can be assembled following receptor activation o These complexes can be assembled on phosphorylated phosphoinositide lipids

• The nature of signaling pathways: Intracellular signaling molecules : signaling complexes o These complexes can be ▪ Organized around a “scaffold” protein ▪ Assembled following receptor activation ▪ Assembled on phosphorylated phosphoinositide lipids
• Receptors and their response to signals o Enzyme-coupled receptors: Receptor Tyrosine Kinases ▪ Activation of RECEPTOR TYROSINE KINASES occurs after ligand binding through dimerization and cross-phosphorylation
• Signaling through Receptor Tyrosine Kinase o Proteins with SH2 domains then bind the phosphotyrosine residues and lead to activation of signaling proteins. o One of these signaling proteins can be Phospholipase C ▪ Ca2+ release likely also mediates other effects not shown here! ▪ Note that this downstream process can be linked to some other upstream event (doesn’t HAVE to be RTK)
• Signaling through G-protein Coupled Receptors o GPCRs are the most common drug targets o G = Guanosine-5'-triphosphate (GTP) o GPCRs are bound to heterotrimeric G-proteins o Without Ligand = Inactive GPCR & G protein bound to GDP o With Ligand = Active GPCR & G protein bound to GTP o Upon activation, bg subunits are separated from the a subunit
• The Role of Calcium as an Intracellular Messenger o Ca2+^ ions have a key role in a remarkable variety of cell activities: ▪ muscle contraction, ▪ cell division, ▪ secretion (exocytosis), ▪ endocytosis, ▪ fertilization, ▪ synaptic transmission, ▪ metabolism, ▪ cell movement
• Calcium ion concentration in the cytosol increases upon activation of ligand-gated Ca2+ ion channels in the ER by the second messenger inositol triphosphate. Lecture 19 11/06/
• Replication is semi-conservative o Each DNA strand is used as a template for the synthesis of a complementary strand

o Damaged DNA, if unrepaired, can persist through cell divisions. o Errors in copying, if unrepaired, are propagated through cell divisions

• Replication occurs in the 5’ to 3’ direction o New nucleotides are added at the 3’ end. o Chain growth occurs in the 5’ to 3’ direction. o Addition of deoxynucleotides requires a primer. ▪ (telomeres are the exception) o This mode of replication results in the antiparallel double helix structure. o DNA replication is catalyzed by DNA polymerase – an enzyme with fingers , palm and thumb domains. o 5’ to 3’ polymerization occurs with an error rate of 1 in 105 nucleotides. Other processes reduce the error rate further.
• Solutions for the 1 st^ three problems of replication o (1) Strand polarity ▪ All synthesis is 5’ to 3’. Okazaki fragments are synthesized on the lagging strand. DNA Ligase seals gap between successive fragments. o (2) Unzip DNA ▪ A hexameric complex called DNA helicase unzips the DNA. It uses ATP and acts as a rotary engine. Unzipped DNA is stabilized by single-stranded DNA binding protein. o (3) Processivity ▪ A sliding clamp holds the DNA polymerase in place. The clamp is loaded on DNA by a clamp loader that uses ATP hydrolysis to lock the clamp around DNA.
• At the replication fork, the polymerases on both the leading strand and the lagging strand are moving in the same direction.
• Rapid rotation of DNA is needed ahead of the replication fork o Problem (4): How to untangle the DNA? o Solution: Topoisomerases relieve torsional stress ▪ Changes the topology of DNA
• Topoisomerase I: nick and swivel mechanism o Topoisomerase I does not require ATP to relieve strain
• Topoisomerase II: gating mechanism o Topoisomerase II requires ATP to untangle DNA. o The enzyme attaches covalently to both strands of one DNA helix, creating a gap in the DNA helix. o The other DNA helix is then passed through the gap.
• What do the enzymes topoisomerase I and topoisomerase II have in common? They both have nuclease activity
• DNA replication begins at replication origins o A region of high AT content
• Problem (5): One copy – Prokaryotic solution o Prokaryotic origins have a refractory period o Methylation is mediated by the Dam methylase in prokaryotes
• What about in eukaryotes? It’s more complex…