BIO130 Section 2

W7-12 + Labs

ECM

Extracellular matrix - specialized material outside of cell

Lysosome

Membrane-bound organelle, made of enzymes and responsible for degradation of cell components that are no longer needed

Lysosome and ECM

specific to animal cells

Cell wall, vacuoles, and chloroplasts

specific to plant cells

Cytoplasm

contents of cell outside nucleus

Cytosol

aqueous part of cytoplasm

Lumen

inside of organelles

Cellular functions occurring at membranes

compartmentalization, biochemical scaffolding, transport of solutes, selective permeability, response to external stimulus, cell interactions

Membrane bilayer components

2 lipid molecule “leaflets” + protein molecules

Lipid molecule structure

Hydrophilic or polar head group, and a hydrophilic tail, making it amphipathic

Membrane lipids can be

Phospholipids, sterols, glycolipids

Polar head group of a phospholipid

Different top group, phosphate, glycerol

Tail of a membrane lipid

nonpolar hydrophobic hydrocarbon chains (CH2) 14-24 hydrocarbons long

Lipid tail kink structure

One saturated chain, one unsaturated with a kink: cis-double bond causing bend

Membrane bilayer formation

In aqueous solutions, phospholipids spontaneously self-associate into a bilayer. polar head interacts with water, hydrocarbon tails interact with each other

Shape of bilayer

energetically favorable and polar heads shield tails from water

Liposomes

artificial lipid bilayers used to study lipid and membrane protein properties, and function for drug delivery into cells

Cell membrane fluidity

Can be deformed without causing damage, but carefully regulated as important

Live cell imaging

laser tweezers are used to manipulate membrane

functions of membrane fluidity

membrane proteins transport, enzyme activity, and signaling

Phospholipid movement in bilayer

rapidly diffuse, rotate laterally, and flex, but rarely flip-flop from one leaflet to another

Factors affecting membrane fluidity

temperature and composition

Lower temperature membrane

more viscous (thick), less fluidity

Composition of membrane for fluidity

more unsaturated phospholipids increase fluidity, shorter tails increase fluidity at lower temperatures, addition of cholesterol stiffens cell membrane

Sterols and cholesterol

both in plants, only cholesterol in animals - stiffens and makes membrane less permeable to polar molecules

Phospholipid translocation

flipping of a phospholipid from one leaflet to the other, necessary because phospholipids are synthesized in the cytosolic leaflet of the endoplasmic reticulum

Scramblases

enzyme catalyzing random and rapid phospholipid translocation in membrane; constantly active in ER

Asymmetry and orientation of lipid bilayer

cytosolic and noncytosolic faces. glycolipids and glycoproteins on noncytosolic face create asymmetry

Flippases

catalyze rapid translocation of specific phospholipids in Golgi membrane to the cytosolic leaflet

specific binding at membrane example

phosphatidylserine binds to cytosolic protein kinase C at plasma membrane

Glycolipids and glycoproteins

formed by adding sugars groups to lipids/proteins on luminal face of Golgi; end up on plasma membrane and in some organelles, provide protection to membrane from harsh environments

components of the plasma membrane

lipid bilayer, transmembrane glycoproteins, cholesterol, oligosaccharides linkers, GPI-linked glycoproteins, glycolipids

Types of membrane proteins

transmembrane, monolayer-associated, lipid-linked, protein-attached

Integral membrane proteins

transmembrane, monolayer-associated, and lipid-attached; inserted in membrane or directly attached to a lipid, extracted with detergents that destroy the bilayer

Peripheral membrane proteins

protein-attached membrane proteins; on either face of membrane, bound to other proteins or lipids with non-covalent bonds; gentle extraction that does not destroy bilayer

Transmembrane protein properties

Amphipathic, hydrophilic domains have polar AA side chains while hydrophobic membrane-spanning have non-polar A side chains

Examples of membrane-spanning domains

around 20 alpha helices, multiple alpha helices, beta barrels

Transmembrane protein function types

Transporters, channels, anchors, receptors, enzymes

Transporter/channel structures + examples

multiple alpha helices; Na+-K+ pump, K+ leak channel

Anchor protein structures + example

alpha helix; integrins

Receptor protein structure + example

single alpha helix; receptor kinases

transmembrane enzyme structure + example

multiple alpha helices; adenylyl cyclaces

x-ray crystallography

technique used to identify 3D structures of transmembrane proteins

Hydrophobicity plots

graph predicting 3D structure of transmembrane proteins using only amino acid number.

Monolayer-associated membrane proteins + example

anchored on cytosolic face by an amphipathic alpha helix, example Sar1 for membrane bending

Lipid-Linked Membrane Protein structures and functions

protein with a GPI anchor does synthesis in ER lumen or end up on cell surface (noncytosolic face); protein with other lipid anchor have cytosolic enzymes to attach anchor and direct to cytosolic face

FRAP

Fluorescence Recovery After Photobleaching, a technique to study protein movement; protein fused to green fluorescent protein (GFP) or labelled, photobleach an area, then in recovery other fluorescent proteins migrate in

Rate of Fluorescence Recovery

Time taken for neighboring unbleached fluorescent proteins to move into bleached area

Artificial bilayer (liposome) permeability

impermeable to most soluble water molecules

facilitated transport

membrane transport proteins facilitate transport of specific molecules through cell membrane

Down a concentration gradient means

high to low concentration

movement of permeable molecules across lipid bilayer

occurs by simple diffusion

Permeable molecules

Hydrophobic or non-polar molecules, including small non polar molecules or small uncharged polar molecules

movement of impermeable molecules across lipid bilayer

requires membrane proteins for transport

Impermeable molecules

ions and large uncharged polar molecules

examples of small nonpolar molecules (permeable!)

O2, CO2, N2, steroids, hormones

examples of small uncharged polar molecules (permeable!)

H20, ethanol, glycerol

examples of larger uncharged polar molecules (impermeable!)

amino acids, nucleosides, glucose

examples of ions (impermeable!)

H+, Na+, K+… anything with a charge

Transmembrane transport proteins

create a protein-lined path across cell membrane to transport specific classes of polar and charged molecules

2 main classes of membrane transport proteins

channels and transporters

Channel protein selectivity and transport

selects by size and charge, transient interactions during transport through open channel; no conformational changes occur

transporter selectivity and transport

solute fits into binding site with specific binding, and conformational changes do occur for transport

passive transport

down concentration gradient (from high to low concentration), not requiring ATP

active transport

against concentration gradient (low to high concentration), does require ATP

Electrochemical/electrical gradient

Concentration gradient + Membrane potential; can be additive or work against each other

Passive transport proteins

Channel proteins and uniports

Channel Proteins

Have a hydrophilic pore across membrane, facilitate passive transport of specific ions based on size and charge with transient interactions with cell wall; faster than transporters

Ion channels

found in plant, animal, and micro-organisms; non-gated (always open) or gated (signal opens)

Types of gated ion channels

Mechanically, intracellular ligand, extracellular ligand, and voltage gated channels

Uniport

transporter protein facilitating reversible transport of one type of solute down the electrochemical gradient

GLUT

uniporter transporting glucose down concentration gradient

Active Transporters

Gradient-driven pumps, ATP-driven pumps, light-driven pumps

Gradient-driven pump

active transporter moving one solute down its electrochemical gradient, using the energy to transport second solute against it

ATP-driven pump

active transporter using ATP hydrolysis to move chemical against its gradient

light-driven pump

bacterial active transporter using light energy to move solute against gradient

Gradient-driven pump types

Symport, moving 2 solutes in same direction; Antiport, moving 2 solutes in opposite directions

examples of symport and antiport

Na+-glucose symport, Na+-H+ antiport

Types of ATP-driven Pumps

P-Type pump, V-type proton pump, ABC transporter

P-type Pump

ATP-driven pump, phosphorylated; important to generate and maintain electrochemical gradients

Na+-K+ pump

ATP-driven P-type pump; phosphorylates and de-phosphorylates; moves 3 Na+ and 2 K+ against gradient.

P-type pumps in animal or plant PMs

Na+-K+ in animal cells, H+ in plant cells, are pumps important for generating electrochemical gradients and membrane potential

ABC Transporter

ATP-Driven pump using 2 ATP to pump small molecules across membrane

V-Type Proton Pump

ATP-Driven pump using ATP to pump H+ into organelles to acidify lumen; plant vacuoles in lysosomes

F-Type ATP Synthase

structurally related to V-Type pump, but pumps opposite direction; in mitochondrion, chloroplasts, and bacteria

Transporters and the intestine relationship

asymmetric transporter distribution on membrane to transfer glucose from intestine to bloodstream

How are transporter proteins restricted and spaced in epithelial intestinal cells

restricted by tight junctions; Na+-glucose symporter on apical membrane; GLUT2 uniporter and Na+-K+ pump on basolateral plasma membrane

Membrane Potential and its uses

Difference in electrical charge on two sides of membrane; Used by gradient-driven pumps for active transportand important for electrical signaling

DNA Isolation

the process of extracting DNA from cells or tissues so that it can be studied or used in experiments. This involves breaking open the cells, removing proteins and other cellular components, and purifying the DNA.

importance of dna isolation

allows scientists to study genes, perform genetic testing, clone genes, sequence genomes, diagnose diseases, and conduct forensic analyses

what plays a major and minor role in generating membrane potential (animal cells)

major: K+ Leak channel, minor: Na+-K+ pump

What helps form membrane potential in plant cells

H+ pump

steps of dna isolation

cell lysis, removal of proteins, precipitation of DNA, washing and resuspension

importance of buffer in DNA isolation

minimizes fluctuations in pH

agarose gel electrophoresis

molecules are placed in wells and separated by an electric current and migrate according to its charges, while its migration rate varies based on shape, charge-to-mass ratio, and size

what is SYBR Safe used for

stains nucleic cid to be viewed under UV light

PCR steps

denaturation, annealing (base pairing), synthesis (extension)

temperature in PCR process

strands are split during denaturation by heating and annealed by cooling, then the temperature is raised to around the middle of these two again for synthesis. these 3 steps are repeated 20-40

mitochondria function

ATP synthesis

Golgi apparatus function

receives proteins and lipids from ER, modifies them, and dispatches them to other places in the cell