Topic 2: Organelles and the Nucleus

Question 1

What is the function of eukaryotic organelles?

Answer 1

eukaryotic cells are composed of many different membrane bound compartments called organelles each of distinct functions

larger eukaryotic cells need a way of maintaining adequate concentrations of reactants (substrates) and catalysts (enzymes) for proper functioning of cellular activities

internal membranes allowed more space and seperation

specificity of function is achieved by differential localization of proteins

Question 2

What is compartmentalization?

Answer 2

organelles provide specificity of function and also concentration of reactions

Question 3

What is the mitochondria?

Answer 3

double-membrane bound: one membrane is eukaryotic in origin, inner membrane is prokaryotic

contains own DNA: encodes some mitochondrial genes, most mitochondrial genes are nuclear encoded

mitochondrial genes are stored in the nucleus for protection

Question 4

What is the endoplasmic reticulum?

Answer 4

a complex network of tubular membranes and flattened sacs (cisternae)

internal space of the ER is called the lumen

continuous with the other membrane of the nuclear envelope

Question 5

What is rough endoplasmic reticulum?

Answer 5

covered in ribosomes that sit on cytoplasmic face

bound ribosomes translate proteins destined for within endomembrane system

Question 6

What is smooth endoplasmic reticulum?

Answer 6

no association of ribosomes

involved in synthesis of lipids, detoxification, Ca++ storage

Question 7

What is the golgi body?

Answer 7

processing and packaging of proteins

site of secretory protein packaging and synthesis of polysaccharides

receives protein from the ER in vesicles (budded compartments)

once produced in the golgi, these proteins can then be passed along to other cellular compartments and including the plasma membrane

modifies cargo by glycosylation and other methods

ships cargo to other destinations within endomembrane system

Question 8

What are lysosomes?

Answer 8

digestive organelle of the cell

contains hydrolytic enzymes that digest only macromolecules (peptidase, DNAse, lipase, sucrase)

enzymes are highly specific to lysosome

Question 9

What are ribsosomes?

Answer 9

protein synthesis

smallest organelle found in cells (30 nm in diameter)

composed of two subunits that differ in both size and shape

most numerous cell organelle

all translation begins in cytoplasm on free ribosomes (except the few mitochondrially translated proteins)

Question 10

What are the plasma membrane?

Answer 10

semipermeable phospholipid bilayer

two membranes (or lipid bilayer) surrounds the cell and its organelles

defines the boundaries of the cell and retains its intracellular contents

consists of phospholipids, other lipids, proteins, and carbohydrates

each phospholipid consists of two hydrophobic tails (hydrocarbon) and a hydrophilic head (phosphate containing); amphipathic

phospholipids spontaneously form a bilayer due to: geometry (columnar shaped) and chemistry (amphipathic)

Question 11

What is centrifugation?

Answer 11

when a particle is subject to centrifugal force, its rate of movement through a solution depends on the particles size and density

Question 12

What is sedimentation rate?

Answer 12

rate of movement through a solution, larger or denser particles have a higher sedimentation rate

Question 13

What is subcellular fractionation?

Answer 13

using centrifugation to isolate and purify organelles and macromolecules based on their sedimentation rates

Question 14

What is a centrifuge?

Answer 14

consists of a rotor that can be spun rapidly by a motor

Question 15

How are sample prepared?

Answer 15

tissues/cells are first homogenized or disrupted to separate contents

sample forced through narrow orifice, ultrasonic vibration, osmotic shock, enzymatic treatment, manual grinding

important to keep organelles and macromolecules intact (often homogenization is performed in cold isotonic solutions)

Question 16

What is differential centrifugation?

Answer 16

supernatant is saved and spun again

Question 17

What is equilibrium or density gradient centrifugation?

Answer 17

add homogenate to a pre-established gradient, you get refined and pure fractions

Question 18

What are the different parts of the nucleus?

Answer 18

1. Nucleoplasm: fluid of nucleus
2. Nuclear Envelope: 2 lipid membranes, outer membrane is continuous with the ER
3. Euchromatin: loosely packed DNA
4. Heterochromatin: tightly packaged DNA
5. Nucleolus: site of RNA transcription and ribosomal assembly

Question 19

What are nuclear pores (NPC)?

Answer 19

nuclear pores span the nuclear double membrane

composed of proteins called nucleoporins arranged in a very large complex

small ions, metabolites and small globular proteins can diffuse freely

other molecules must be actively imported/exported through nuclear pore

Question 20

What is the structure of the nuclear pores?

Answer 20

composed of 465 individual proteins

cytoplasmic side contains cytoplasmic filaments

pore complex spans both bilayer (octagonal pore)

transport is non-selective for anything smaller than 40 kDa

nuclear basket faces nucleoplasm

Question 21

What is nuclear import?

Answer 21

all proteins start translation in the cytoplasm therefore they must be imported into the nucleus

nuclear proteins such as: ribosomes, DNA polymerase, RNA polymerases, histones, etc. must all be imported into the nucleus by a specific mechanism

all nuclear-destined proteins contain a nuclear localization signal (NLS)

Question 22

How are proteins targeted in the nucleus?

Answer 22

the nucleus requires many proteins that are all synthesized in the cytoplasm to enter and exit in a regulated fashion

most of the molecules needed inside the nucleus are impermeable to the double membrane and must pass through nuclear pores

Question 23

How does a protein “know” it is supposed to go to the nucleus?

Answer 23

NLS = Lys-Lys-Lys or Arg-Lys-Lys/Arg

Question 24

What is the necessity experiment for NLS?

Answer 24

take it away and see if it still works

SV40T antigen = normally nuclear b/c has NLS

NLS was genetically deleted

reintroduced into cell culture

stained with immunofluorescence

protein was not nuclear localized

NLS is necessary for nuclear localization

Question 25

What is the sufficiency experiment for NLS?

Answer 25

pyruvate kinase is an enzyme that normally localizes to the cytoplasm genetically engineered an NLS into a normally cytoplasmic protein NLS-PK fusion protein localizes to nucleus therefore NLS is sufficient for localization

Question 26

What is the mechanism of import into the nucleus?

Answer 26

cytoplasmic proteins called importins are responsible for recognizing and binding the NLS importins are dimers of alpha and beta subunits forming a heterodimeric nuclear-import receptor alpha subunit binds NLS, beta subunit binds NPC (cytoplasmic filaments of nuclear pore complex entire nuclear import receptor: cargo (NLS containing protein) is moved across NPC into nucleus inside nucleoplasm the NIR cargo complex interacts with Ran-GTP and allows dissociation G proteins: a large class of small proteins that reversibly bind to GTP

Question 27

What are the steps of import into the nucleus?

Answer 27

1. NLS containing protein = fully folded, translated 2. NIR (receptor) binds NLS protein 3. Translocation into nucleus (localization of Ran-GEF in nucleus allows formation of Ran GTP) 4. Cargo Dissociation (Ran GTP binds NIR and causes release of cargo into nucleus) 5. NIR and Ran GTP leave nucleus 6. Ran GTPase activity in cytoplasm causes Ran GTP --> Rand GDP (Ran hydrolyzes it bound GTP, Ran GDP dissociates from receptor)

Question 28

What is nuclear export?

Answer 28

similar to import except now a group of exportins is used proteins that shuttle in and out of the nucleus must have a Nuclear Export Signal (NES) ribosomes would have both NLS and NES

Question 29

What are the steps of nuclear export?

Answer 29

exportin recognizes and binds NES entire exportin-Ran-NES complex leaves through NPC Ran GAP in cytoplasmic filaments hydrolyzes Ran GTP --> Ran GDP therefore causes exportin to release cargo

Question 30

What is the differences between import and export to the nucleus?

Answer 30

in export the Ran GTP is part of the cargo complex that is transported

Question 31

Why doesn't import and export to the nucleus get mixed up?

Answer 31

same Ran protein exportin and importin are differentially regulated by Ran Ran GDP = in cytoplasm, Ran GTP = in nucleus exportin works if bound to cargo AND Ran GTP importin works if bound to cargo OR Ran GTP

Question 32

What is DNA packaging?

Answer 32

nuclear genetic material of eukaryotic exists in two states: interphase chromosome: cell is not dividing, majority of the time condensed chromosome: during cell division

Question 33

What is euchromatin?

Answer 33

loosely packaged DNA that is transcriptionally active, light staining regions

Question 34

What is heterochromatin?

Answer 34

tightly packages DNA that is inaccessible to transcription, dark staining regions

Question 35

What are nucleosomes?

Answer 35

1st order of chromatin packaging a nucleosome consists of: histone octamer of H2A, H2B, H3, and H4 positive charge on histone interacts with the negative of the DNA backbone, therefore not sequence specific interactions

Question 36

What are histones?

Answer 36

highly conserved histone H1 is less conserved but not part of core nucleosome histone H3 has a tail of amino acids = site of regulation histone tails can be reversibly phosphorylated, methylated or acetylated which changes degree of packaging methylation methylation = closes chromatin = less transcriptionally active = heterochromatin acetylation = opens chromatin = more transcriptionally active = euchromatin

Question 37

What is higher order packaging?

Answer 37

solenoid or 30 nm filament coiling of nucleosome filament into higher-order, thicker filament zig-zag ribbon structure that is wound into a helix

Question 38

What are non-histone scaffolding proteins?

Answer 38

the 30 nm fiber is then packaged further into looped domains by some DNA associating with non-histone scaffolding proteins highly organized with regions of DNA associating together in a predictable way

Question 39

What are mitotic chromosomes?

Answer 39

fully condensed therefore transcriptionally active

Question 40

What is nuclease?

Answer 40

an enzyme that digests DNA by breaking phosphodiester bonds 2 types: exonuclease (pac-man), endonuclease (scissors) endonuclease can only digest DNA in linker regions because the DNA is associated with histones

Question 41

What is an experiment with endonuclease?

Answer 41

uncoil DNA expose to low concentrations of endonuclease )ensures random cuts) run DNA on a DNA gel (separates DNA based on size) low concentrations of endonuclease gives pattern of band sizes that represent multiples of nucleosome + linker length therefore linker is short at higher concentrations only DNA protected in nucleosome is intact