BIO130 - Week 7 Lecture + Textbook

The specialized material outside of the cell

Found only in animal cells

What is the Extracellular Matrix? In what organisms is it found?

An organelle that manages the degradation of unnecessary cellular components

What is a Lysosome? In what organisms is it found?

The wall surrounding the cell, protecting against mechanical stress and providing the cell with its shape

Found in plant cells only

What is the Cell Wall? In what organisms is it found?

‘Sacs’ - 2 types

• Degradation (similar function to animal lysosome)

• Storage (small molecules, proteins)

Found in non-animal cells

What is the Vacuole? In what organisms is it found?

The organelle at which photosynthesis occurs

Not found in animal cells

What is the Chloroplast? In what organisms is it not found?

Cytoplasm: The contents of the cell outside of the nucleus, including the cytosol

Cytosol: The aqueous part of the cytoplasm

Lumen: The inside of organelles

Lumen + cytosol = cytoplasm!

Distinguish between the Cytoplasm, Cytosol, and Lumen in eukaryotes

1. Compartmentalization

2. Scaffolding for biochemical activities

3. Selectively permeable barrier

4. Transportation of solutes

5. Response to external signals

6. Intercellular interactions

What are some functions that occur at cellular membranes in eukaryotic cells?

A two-layer (or ‘leaflet’) sheet of amphipathic phospholipids and proteins

What is a Membrane Bilayer?

Hydrophilic/polar ‘head’ group, facing water

Hydrophobic tails, ‘tucked away’ into the inner part of the bilayer

What two parts make up a phospholipid?

Phospholipids, Sterols, Glycolipids

What are the different kinds of lipids found in membranes?

A specific type of lipid containing a phosphate group within the polar head

Many contain a glycerol group, giving them the name phosphoglycerides

Structure:

• <Various groups>

• Phosphate

• Glycerol

• Hydrocarbon tails

Explain the structure of phospholipids - What are their defining features? What is the overall structure?

A phospholipid with a phosphate group attached to a glycerol

The glycerol then connects the two hydrocarbon tails

What is a phosphoglyceride?

14-24 carbon atoms long

Typically have one saturated (all C-C bonds), one unsaturated (cis C=C bond, forming a ‘kink’) tail

Explain the features of the phospholipid hydrocarbon tails.

• Most energetically favourable arrangement

• Polar head group interacts with water (favourable)

• Hydrophobic hydrocarbon tails interact with other hydrocarbon tails (favourable)

Why do phospholipids spontaneously self-associate into a layer in an aqueous environment?

More energetically favourable - in a planar bilayer, hydrophobic tails will be exposed to water along the edges

This does not occur in a sealed compartment, shielding the tails from water

Why do phospholipid bilayers assemble into sealed compartments?

An artificially created lipid bilayer, used to study lipid properties, membrane protein properties, and drug delivery into cells

Closed, sphericle vesicle of pure phospholipids - always artificial

What is a Liposome?

No, they are fluid and can be deformed without damaging

Are cell membranes solid?

• Lipids, within each leaflet, can

  • Diffuse laterally (sideways movement)

  • Rotate (rotate horizontally)

  • Flex (flex hc tails)

• Phospholipids can move between layers (flip-flop), but do so rarely due to energetic unfavourability

  • Exposure of hydrophobic to water

How can phospholipids move? Explain.

No, it is carefully regulated as it is important for function (i.e. membrane proteins used for transport, signaling, etc.)

• Rigidity would inhibit function, but so would lack of regulation

Is cell membrane fluidity freeform and unregulated? Why or why not?

• Temperature (lower temp → less fluid, more viscous)

• Phospholipid saturation (more unsaturated hc chains → increase fluidity, reduce tight packing) at lower temperatures

• Tail length (shorter tails → less interaction, more fluid) at lower temperatures

• Lipid composition (addition of cholesterol → stiffens membrane, lowers permeability)

What factors can affect membrane fluidity? How so?

Animal: primarily Cholesterol

Plant: More versatile plant sterols, some cholesterol

What is the most frequently seen cholesterol in animal cells? What about Plant cells?

• Decreases mobility of phospholipid tails by inserting itself and stiffening membrane

• Decreases permeability of polar molecules, allowing more control over entry

What is the maximum ratio of cholesterol and phospholipids? What is the affect of adding more phospholipids?

• Scramblase

• Catalyzes the rapid flip-flop of random phospholipids between leaflets

• Necessary due to phospholypid synthesis in the cytosolic leaflet of the ER, which causes asymmetry unless random phospholipid flip flop occurs

What enzyme catalyzes the movement of phospholipids between leaflets? What are the properties of this switch? Why is this needed?

False - a given face will always remain on the same side as it travels (i.e. a protein on a cytosolic face will always be facing the cytosol)

T/F: Membranes switch orientation as they move between organelles

Enzymes in the Golgi membrane flipping lipids between leaflets

Flippase

• Catalyzes the rapid flip-flop of specific phospholipids to the cytosolic leaflet

• No flippase that can move cytosolic → lumen, only the other way around

Some types of lipids can bind cytosolic proteins at the plasma membrane

How does asymmetry in the lipid bilayer occur?

• Proteins and lipids with sugars added on the luminal face of the golgi

What is a Glycolipid/Glycoprotein?

• Typically end up on the noncytosolic face of the plasma (external) membrane or the inside of some organelles

• Protect the membrane from harsh environments (e.g. high pH)

Where do glycoproteins/lipids typically end up? What is their function?

• Transmembrane

• Monolayer-Associated

• Lipid-Linked

• Protein-Attached

What are the four kinds of membrane proteins?

A membrane protein directly attached to the lipid bilayer

• Transmembrane, Monolayer-associated, Lipid-linked

• Can be inserted into a bilayer themselves, or attached to a lipid inserted into the bilayer

What is an Integral Membrane Protein? What are the types of proteins in this category? How are they extracted from the bilayer?

Proteins that do not directly insert into the membrane

• Typically bound to other proteins or to lipids by non-covalent interactions

Gentle extraction methods which leave the lipid bilayer intact are used

What is a Peripheral Membrane Protein? How are they extracted?

Amphipathic proteins that cross the entire membrane and emerge out of either side

• Have both hydrophilic domains within the aqueous outside (AA side chains are polar) and hydrophobic domains within the membrane (non-polar AA)

Explain the domains of transmembrane proteins

• Single-pass alpha helix

  • Alpha helix passing vertically through the phospholipid layer

• Multipass alpha helix

  • Multiple alpha helices arranged such that the hydrophilic side chains form an aqueous pore

• Rolled beta sheet (Beta-barrel)

  • Meshwork, open channel made of beta sheets

What are the three types of Transmembrane proteins?

~20 hydrophobic amino acid side chains

What is the typical length of a membrane spanning alpha helix?

X-Ray Crystallography

Hydrophobicity Plots

What are the ways in which transmembrane protein structures are identified?

• Determination of a protein’s 3-D structure

• An X-ray is shined onto a protein crystal, getting a unique diffraction pattern - using this pattern, the structure is calculated

Explain X-Ray Crystallography.

Segments of 20-30 hydrophobic amino acids can span the lipid bilayer as an alpha helix

• By observing a protein’s AA composition, we can measure each amino acids hydropathy index.

  • Small, sharp points of +delta G will likely be the amino acids within a hydrophobic domain

  • The number of these sharp regions is the umber of alpha helices (single/multipass)

Explain Hydrophobicity plots

Attached to cytosolic face by an amphipathic alpha helix (part of helix is exposed to aqueous cytosol, part is not)

Example: Proteins used in bending membranes to form vesicles at the ER make use of MAMP

What side are Monolayer-Associated Membrane Proteins attached? What are some examples of this?

Two types:

• A protein with a GPI anchor

  • Synthesized on ER lumen, ends up on noncytosolic face of cell surface

• Protein with a non-GPI lipid anchor (i.e. fatty acids0

  • Anchor added by cytosolic enzymes

  • Protein is directed to a cytosolic face

What is a Lipid-Linked Membrane Protein? What are the two types of this? Where do they end up?

• A detergent is added to a membrane with a protein

• The amphipathic detergent monomers form large ‘micelle' spheres reversibly

• In doing this in the presence of the membrane, it splits the protein and lipid into micelle groups

Explain the process of extracting membrane proteins using detergent

1. Detergent micelles + monomers are added to a cell membrane, solubizing the proteins and lipid into separate groups

2. The protein of interest is purified from the rest of the solution

3. The detergent-phospholipid micelles are added to the protein, and the detergent micelles + monomers are removed

4. A new, artificial bilayer is formed, and the protein is incorporated

List the process for transferring proteins into an artificial bilayer

• F.R.A.P

• Fluorescence Recovery After Photobleaching

How can lateral diffusion/study of protein movement be measured in membranes?

• Protein of interest is fused to G.F.P (Green Fluorescent Protein)

• An area of the cell is photobleached to white

• The rate of colour recovery can be used to estimate the rate of diffusion in a cell

  • Recovery comes from neighbouring, unbleached proteins randomly moving around and diffusign into the area

Explain Lateral Diffusion of Membrane Proteins

• The time taken for neighbouring, unbleached proteins to move into a bleached area

  • Can go to full, or not

What is the Rate of Fluorescence Recovery?

Yes, via tight junctions

• Called Membrane Domains

Can membrane protein movement be inhibited? What are the ranges of uninhibited movement called?

A protein-studded film, invisible to light microscopes

• Protein bilayer

• Selective channels

Define a Plasma Membrane

Bacteria: 1 (external)

Eukaryotes: Multiple, external + internal membranes

• Enclose cell, ER, Golgi, endosomes, mitochondria

How many plasma membranes do bacteria have? Eukaryotes?

Organic solvents - insoluble in water

What are lipids soluble in?

Amphipathic

What is the most common property of membrane lipids?

An abundant phospholipid - choline attached to a phosphate group as a hydrophilic head

What is Phosphatidylcholine?

~25 nm

What is the minimum diameter of a vesicle?

~20% by weight on average

What percent of a plasma membrane is made up of cholesterol in animal cells?

• Allows membrane proteins to rapidly diffuse and interact

• Permits membrane lipids and proteins to diffuse from site of insertion to other regions

• Ensures even distribution of membrane during division

• Allows fusion of membranes

What other reasons is fluidity important in a cell?

Cytosolic surface of ER

Where are phospholipids manufactured in eukaryotes?

Golgi apparatus, with the flippases

Where does asymmetry first arise in the membrane?

Cytosolic monolayer, noncytosolic monolayer

What are the two lipids of the bilayer?

50%

What percentage, by mass, of an animal cell membrane is proteins?

Small, lipid-like, often cone-shaped molecule with one hydrophobic tail

What is the typical shape of detergent molecules?