BIO130 Final Exam

A detailed set of flashcards on information tested on in the final exam (second half of the course + labs)

What are the two things animal cells have but plant cells don’t?

Lysosomes → degradation

Extracellular matrix

What are the three things plant cells have but animal cells don’t? And what are their purposes?

Cell wall → cell shape, protection against mechanical stress

Vacuoles (2 types) → degradation (like animal lysosome), storage

Chloroplast → photosynthesis

Cytoplasm vs Cytosol vs Lumen

Cytoplasm: everything but nucleus (includes organelles)

Cytosol: everything but membrane bound organelles (aqueous part of cytoplasm)

Lumen: inside of organelles

3 types of lipids that compose membranes

phospholipids, sterols, glycolipids

phosphoglyceride and structure

type of phospholipid with glycerol group

• glycerol group: 3 C with O connected. 2 Os connects to the hydrocarbon tails, the other connects to the phosphate group.

liposome

artificial lipid bilayer in sphere (because that’s energetically favorable)

Uses:

• drug delivery into cells

• study lipid and membrane protein properties

Describe the ways phospholipids can move (fluid)

Phospholipids rapidly move within each leaflet

• lateral diffusion (side to side, deeper into plane)

• rotate

• flex

• and rarely “flip flop” - move from one leaflet to another on their own

effect of temperature on membrane fluidity

lower temperature → more viscous, less fluid

factors affecting membrane fluidity (membrane composition)

1. phospholipid saturation

   1. cis-double bonds increase fluidity (reduce tight packing)

   2. phospholipid tail length (shorter tails increase fluidity because lipid tails interact less)

   3. lipid composition (addition of cholesterol stiffens membrane)

cholesterol

most common sterol in animal membranes.

Decreases mobility of phospholipid tails, makes plasma membrane less permeable to polar molecules.

scramblase

aka “Phospholipid translocator”

Enzyme in the ER that catalyzes the flip-flopping of random phospholipids from one leaflet to the other.

Why is scramblase needed?

Phospholipids are synthesized in cytosolic leaflet of ER which means the membrane needs to be evened out.

Flippase

The two faces of the plasma membrane have different lipids. This asymmetry is maintain by the enzyme Flippase in the Golgi membrane.

• flip flops specific phospholipids to the cytosolic leaflet

4 steps to new phospholipids being made and added to the cell membrane

1. in ER membrane you make a ton of lipids

2. there’s some scramblases that flip them to other side to even things out

3. transport vesicles takes it to Golgi

4. Flippases sorts things out to get specificity —which from then on stays consistent

note: from then on, “stays consistent”

glycolipids/glycoproteins

Formed when sugar groups are added to lipids/proteins on luminal face. Protects the membrane from harsh environments.

integral membrane proteins

membrane proteins that insert in some way into the lipid bilayer

• transmembrane - pass through entire membrane

• monolayer associated

• lipid-linked

peripheral membrane proteins

no not insert into the lipid bilayer. They are associated with the membrane noncovalently, bound to either lipids or other proteins.

properties of transmembrane proteins

amphipathic - AA side chains are either polar or not polar

• the nonpolar region is typically 20-30 hydrophobic amino acids.

Have specific orientations important to function

X-ray crystallography

technique used to determine the 3D structure of proteins

Turns proteins into crystals, light is shown through them, and the diffraction pattern is measured

Hydrophobicity plots

x-axis: AA number. Starts on N-terminus, goes to C-terminus.

high y value = very hydrophobic, low y value = very hydrophillic

• each stretch of 20-30 hydrophobic AAs is a transmembrane domain

Monolayer Associated Membrane Proteins

A type of integral membrane protein

• protein is anchored on cytosolic face by amphipathic alpha helix.

Ex. Sar1 - involved in membrane bending, vesicle formation

lipid-linked membrane proteins

type of integral membrane protein

2 types:

• Protein with GPI anchor on noncytosolic face

• Protein with lipid anchor on cytosolic face

Use of detergent

extraction of membrane proteins

FRAP (Fluorescence Recovery After Photobleaching

Experiment to observe lateral diffusion of membrane proteins.

• you can measure the speed of lateral diffusion by looking at the slope of recovery

membrane transport proteins

2 classes: channel proteins, transporter proteins

Transport polar and charged molecules (small, nonpolar molecules can just simply diffuse on their own)

• selective

channel proteins

Type of membrane transport protein for passive transport

Binds weakly to transported molecule; does not change conformation a lot

Selective for size and charge

Transporter proteins

type of membrane transport protein - can do passive and active transport

Solute binds strongly; conformation changes a lot during transport

membrane potential

the difference in charge across the membrane

electrochemical gradient

concentration gradient + membrane potential (but we take concentration gradient to be more significant)

describe channel proteins in more detail

passive transport

Hydrophilic pore across membrane

Selective for size and charge

Example = ion channel

ion channels and their 2 kinds

passive transport of ions - selective for ion size and charge.

2 kinds:

1. Non-gated Ion Channels - always open

   1. ex. K+ leak channel

2. Gated Ion Channels - signal required to open channel

4 types of gated ion channels

comparison of transporter-mediated diffusion and simple diffusion/channel-mediated transport (rates of transport)

Uniport

Passive transport transporter protein (down electrochemical gradient)

Uni → “one” solute

ex. glucose transporter (GLUT Uniporter)

Gradient driven pump

Type of active transport transporter protein

1 solute down its gradient, 2nd solute against its gradient

ATP-driven pumps (ATPases)

type of active transport transporter protein

Requires ATP hydrolysis to move solute against its gradient

light driven pump

type of active transport transporter protein in bacteria

Describe the 2 types of gradient driven pumps

1. symport

   1. 2 solutes moved in same direction

2. antiport

   1. 2 solutes moved in opposite direction

For both, free energy from 1st solute moving down its electrochemical gradient is used to transport the 2nd solute against its electrochemical gradient.

How is the Na+ electrochemical gradient maintained?

Symports and Antiports are present

Na+ - K+ pump (plasma membrane ATP-driven pump) in animal cells.

How is cytosolic pH regulated?

Antiport: Na+ - H+ exchanger on plasma membrane

What are the three kinds of ATP-driven pumps"?

1. P-type pumps

2. ABC transporter

1. V-type pump

P-type pumps (ATP driven pump)

Generates and maintains electrochemical gradients

• ex. Sodium Potassium pump (3 Na+ out, 2 K+ in)

• ex. H+ pump in plant cell plasma membranes

* “pees itself” → phosphorylated during pumping cycle

(note: another example is Flippase)

why is the Na+ gradient important?

Transport nutrients into cells (e.g. glucose)

maintain pH

How does the Na+ - K+ pump work?

1. 3 sodiums bind

2. the pump phosphorylyzes itself triggering conformational change

3. 3 sodiums ions ejected, 2 potassium ions bind

4. pump dephosphorylates itself, returns to original conformation and the potassium is ejected.

ABC Transporter

type of ATP-driven pump

Uses 2 ATP to pump small molecules across cell membrane

V-type pump

Type of ATP-driven pump

Uses ATP to pump H+ into organelles to acidify the lumen

Found in lysosomes and plant vacuoles

V-type proton pump vs F-type ATP Synthase

They are structurally related but opposite modes of action.

V-type proton pump uses ATP to pump H+ against the electrochemical gradient.

but F-type ATP Synthase uses the H+ electrochemical gradient to produce ATP.

How is glucose transferred from the intestine to the bloodstream?

Lining the gut lumen are epithelial cells, each separated by tight junctions (so glucose can’t go between them).

Na+ driven glucose symporter on the apical domain brings Na+ and glucose into the cell, then Na+ - K+ pump and passive glucose uniport (GLUT Uniporter) on the basal domain remove Na+ and glucose from the cell.

Which transporter proteins are involved with the generation and maintanence of membrane potentials?

K+ leak channel (passive transport)

• outward flow of K+

Na+ - K+ pump (P-type pump)

• Net 1 (+) ion pumped out

^ net result = outside is more + than outside

for plants: H+ pump (P-type pump)

K+ leak channel

Important channel for maintaining membrane potential

Na+ - K+ pump

~10% of membrane potential

• maintains Na+ gradient w low cytosolic [Na+] and K+ gradient with high cytosolic [K+]

Electrogenic:

• 3 Na+ ions pumped out

• 2 K+ ions pumped in

• Net 1 (+) ion pumped out

H+ pump and generation of membrane potential in plant cells

Generates H+ electrochemical gradient, which is used by gradient-driven pumps like H+ Driven Symporter.

• good for electrical signaling and pH regulation

resting membrane potential

When membrane potential is as equilibrium - Voltage difference is steady

• for animals, is from -20 mV to -200 mV

• for plants, is from -120 mV to -160 mV

note: is from perspective of the inside of the membrane

What molecules diffuse rapidly?

small, nonpolar, and sometimes if small enough uncharged polar

ex. O2, CO2

briefly what are the functions of the organelles?

What is cytosol?

50% of the cell volume

Protein synthesis and degradation

many metabolic pathways

contains cytoskeleton

pancreatic exocrine cell

secretes digestive enzymes

rough ER and smooth ER

rough

• has membrane-bound ribosomes

• involved in synthesis of soluble proteins and transmembrane proteins for the endomembrane

smooth

• phospholipid synthesis

• detoxification

Which organelles are not membrane bound?

Nucleolus and Centrosome

Simply, how are proteins sorted?

If they have a sorting signal called a signal sequence they are directed to the right place.

Signal sequence

AA sequence in a protein that directs protein to the correct compartment

• recognized by sorting receptors

encoded in the genome

sorting receptors

recognize signal sequences and take proteins to their destination

2 ways protein sorting is done:

1. post translational sorting

   1. proteins are already fully synthesized in the cytosol before sorting

2. co-translational sorting - for proteins going to ER

   1. have an ER Signal Sequence

   2. proteins complete synthesis on the ER membrane

Post-Translational Protein Sorting

Depending on destination, proteins are either folded or unfolded

• folded: nucleus, peroxisomes

• unfolded: mitochondria, plastids

How does protein sorting into nucleus work?

Post-translational. Protein already folded.

Travel through nuclear pores.

Protein has nuclear localization signal

how are proteins sorted into peroxisomes?

Post translational. Folded.

Proteins imported through transmembrane protein complex.

How are proteins sorted into mitochondrion and chloroplasts

Post translational. Unfolded.

Proteins are unfolded for import by hsp70 chaperone proteins

Co-Translational Protein Sorting

For proteins that enter the ER - the entry point to the Endomembrane system

ER Signal sequence is hydrophobic

Endomembrane system

ER, Golgi apparatus, endosomes, lysosomes

The intracellular compartments exchange lipids and proteins.

Has 3 pathways:

• secretory pathway

• endocytic pathway

• retrieval pathway

In general what are the steps of co-translational sorting?

Translation starts on ribosomes in the cytosol per usual, but then once ER Signal Sequence is translated the protein is inserted into the ER (“co-translational translocation)

The process looks different depending on if the protein is:

1. soluble

2. or transmembrane

Describe the process of co-translational sorting of a soluble protein

SRP recognizes and binds to ER signal sequence once translated, translation stops. This binds to ribosome too.

This complex docks onto SRP receptor on membrane.

SRP leaves and protein synthesis continues with protein transfer into ER lumen.

The ER signal sequence is cleaved by signal peptidase.

Finished. Protein released into ER lumen and translocon closes. The ER signal sequence is eventually degraded.

translocon

A gate for the polypeptide chain to be threaded through to get to the lumen. The ribosome docks on it.

Describe the process of co-translational translocation of a transmembrane protein with N-terminal ER Signal Sequence

Starts the same as for soluble protein, but then a Stop-transfer sequence (internal hydrophobic segment) enters the Translocon.

Protein transfer stops and transmembrane domain is released into the lipid bilayer. Done → signal peptidase cleaves ER signal sequence and Translocon closes.

describe the process of co-translational translocation of a transmembrane protein with an internal ER signal sequence

almost the same as for when there’s a N-terminal ER Signal Sequence but the start-transfer sequence emerges first then the stop-transfer sequence. Transmembrane protein makes two passes at least (threaded through)

note: the start-transfer sequence is not removed

secretory pathway of endomembrane system

Proteins and lipids made in the ER then delivered to other compartments

• Exocytosis = ER to outside

  • via vesicles. Vesicle membranes become part of the plasma membrane

• or ER to lysosomes

2 kinds:

1. constitutive exocytosis pathway

2. regulated exocytosis pathway

Endocytic Pathway of the endomembrane system

contents move into the cell (endocytosis)

• via vesicles. Vesicle luminal contents come from extracellular space. The vesicle membrane is formed from the plasma membrane

Constitutive Exocytosis Pathway (1 type of secretory pathway)

In all eukaryotic cells

Continual delivery of proteins (both transmembrane and soluble) and lipids to the plasma membrane.

Regulated Exocytosis Pathway (1 type of secretory pathway)

Regulated secretion in specialized cells

Solute sorted in specialized secretory vesicles.

An extracellular signal is needed for vesicle fusion with PM and contents to be released.

ex. pancreatic beta-cells → insulin release w increased blood glucose

Golgi Apparatus’s role in the endomembrane system

Receives proteins and lipids from the ER, modifies them, and then dispatches them to other destinations in the cell.

Protein Glycosylation

one of the protein modifications that occurs in the Golgi Apparatus.

In the ER, a single type of oligosaccharide is attaches to many proteins.

Then these oligosaccharides are modified (glycosylation) in the various golgi sacs.

• there’s different enzymes in each cisterna

endosomes and lysosomes

membrane-bound organelles containing material ingested by endocytosis

describe the endocytic pathway

Endocytic vesicles fuse w Early Endosomes and with each other. Ingested material is sorted—either stays in endosomes or recycled into plasma membrane.

Early Endosomes gradually mature into Late Endosomes.

1. moves towards nucleus, stops recycling material to PM

2. Hydrolases and H+ pump are delivered from the trans Golgi network (compartment of Golgi apparatus) by vesicles.

   1. H+ acidifies endosomal lumen

   2. hydrolases become more active

3. Late Endosomes mature into Lysosomes

   1. H+ pumps and Hydrolases continue to be delivered from the Golgi, so pH continues to drop and Hydrolases digest ingested material

   2. Late Endosomes fuse with each other and with lysosomes

4. lysosome = final destination for endocytosed material being digested

How do lysosomes work?

Have ~40 types of hydrolytic enzymes (add water to break down molecules) (ex. proteases, nucleases, lipases)

Lysosomes need to be acidified by H+ pump

It’s membrane bound so protects rest of cell from digestion

• and has glycosylated lysosomal membrane proteins on lumenal side to protect itself from its proteases

Transport proteins in lysosomal membrane transfer digested products (AAs, sugars, nucleotides) to cytosol.

When are signal sequences not removed after arrival to their destination?

Nuclear proteins, because nuclear membranes break down during cell division and the proteins need to “save their ticket” to return.

What are the 3 components of the cytoskeleton?

Actin filaments, Microtubules, Intermediate Filaments

which two components of the cytoskeleton are involved with cell division?

Actin filaments and Microtubules

Which type of microscopy should we use to see the cytoskeleton in detail? Describe how it works.

Transmission electron microscopy

• uses beams of high energy electrons

• resolution limit of ~ 1 nm

Which type of microscopy can be used to determine the location of proteins within a cell? Describe how it works.

Immunofluorescence microscopy

• cells are fixed

• uses a primary antibody that binds to the target protein

• uses a secondary antibody that binds to the primary antibody that is covalently tagged to a fluorescent marker.

• microscope excites the marker.

rank the relative diameters of the three types of cytoskeleton filaments

actin filaments = small

intermediate filaments = intermediate

microtubules = large

What are Intermediate Filaments used for?

structural support

Describe the two types of intermediate filament proteins

Cytoplasmic IFs

• in animal cells subjected to mechanical stress

• provides mechanical strength

Nuclear IFs

• nuclear lamina - 2D meshwork

  • formed by lamins - in all animal cells

Describe the structure of cytoplasmic intermediate filaments

proteins coil together, ropelike - noncovalently:

• monomer = protein with alpha-helical central rod

• 2 monomers = coiled-coil dimer

• 2 dimers = nonpolar staggered antiparallel tetramer

• 8 tetramers = 1 filament (nonpolar)

Keratin Filaments

The Intermediate Filaments in epithelial cells

They are anchored inside each cell at desmosomes (cell-cell junctions) and connect to neighboring cells.

epithelium

sheet of cells covering an external surface or lining an internal body cavity

What are microtubules made of? Describe its structure.

Tubulin - long, stiff hollow tubes

Subunit = two closely related globular proteins

• alpha-tubulin and beta-tubulin that both bind to GTP and to each other to form a polar tubulin heterodimer

13 protofilaments per tube. ← all noncovalent bonding

At which end of a microtubule does growth and assembly occur more rapidly?

+ end

Describe the importance of beta-tubulin.

After it’s been in the protofilament for a while, beta-tubulin cuts GTP to GDP (T-form/GTP heterodimer → D-form/GDP heterodimer). This has weaker binding.

So fast growth means there’s a strong GTP cap.

Microtubule Organizing Centers (MTOCs)

Where microtubules grow out of (has nucleating sites).

• the (-) end is stabilized at the MTOC

ex. centrosome in animal cells

describe how dynamic instability works for microtubules - growth

growth: αβ-tubulin dimers bound to GTPs are added at the (+) end. Shortly after dimer is added, β-tubulin hydrolyzes GTP to GDP. If growth is fast, this hydrolysis is slower than the addition of new dimers so that there’s a strong GTP cap to be added upon.

Describe how dynamic instability works for microtubules - shrinking

GTP hydrolysis is faster than the addition of αβ-tubulin dimers, the GTP cap is lost. The microtubule disassembles due to weaker binding.