Topic 2: Organelles and the Nucleus Flashcards
What is the function of eukaryotic organelles?
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
What is compartmentalization?
organelles provide specificity of function and also concentration of reactions
What is the mitochondria?
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
What is the endoplasmic reticulum?
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
What is rough endoplasmic reticulum?
covered in ribosomes that sit on cytoplasmic face
bound ribosomes translate proteins destined for within endomembrane system
What is smooth endoplasmic reticulum?
no association of ribosomes
involved in synthesis of lipids, detoxification, Ca++ storage
What is the golgi body?
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
What are lysosomes?
digestive organelle of the cell
contains hydrolytic enzymes that digest only macromolecules (peptidase, DNAse, lipase, sucrase)
enzymes are highly specific to lysosome
What are ribsosomes?
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)
What are the plasma membrane?
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)
What is centrifugation?
when a particle is subject to centrifugal force, its rate of movement through a solution depends on the particles size and density
What is sedimentation rate?
rate of movement through a solution, larger or denser particles have a higher sedimentation rate
What is subcellular fractionation?
using centrifugation to isolate and purify organelles and macromolecules based on their sedimentation rates
What is a centrifuge?
consists of a rotor that can be spun rapidly by a motor
How are sample prepared?
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)
What is differential centrifugation?
supernatant is saved and spun again
What is equilibrium or density gradient centrifugation?
add homogenate to a pre-established gradient, you get refined and pure fractions
What are the different parts of the nucleus?
- Nucleoplasm: fluid of nucleus
- Nuclear Envelope: 2 lipid membranes, outer membrane is continuous with the ER
- Euchromatin: loosely packed DNA
- Heterochromatin: tightly packaged DNA
- Nucleolus: site of RNA transcription and ribosomal assembly
What are nuclear pores (NPC)?
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
What is the structure of the nuclear pores?
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
What is nuclear import?
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)
How are proteins targeted in the nucleus?
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
How does a protein “know” it is supposed to go to the nucleus?
NLS = Lys-Lys-Lys or Arg-Lys-Lys/Arg
What is the necessity experiment for NLS?
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
What is the sufficiency experiment for NLS?
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
What is the mechanism of import into the nucleus?
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
What are the steps of import into the nucleus?
- NLS containing protein = fully folded, translated
- NIR (receptor) binds NLS protein
- Translocation into nucleus (localization of Ran-GEF in nucleus allows formation of Ran GTP)
- Cargo Dissociation (Ran GTP binds NIR and causes release of cargo into nucleus)
- NIR and Ran GTP leave nucleus
- Ran GTPase activity in cytoplasm causes Ran GTP –> Rand GDP (Ran hydrolyzes it bound GTP, Ran GDP dissociates from receptor)
What is nuclear export?
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
What are the steps of nuclear export?
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
What is the differences between import and export to the nucleus?
in export the Ran GTP is part of the cargo complex that is transported
Why doesn’t import and export to the nucleus get mixed up?
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
What is DNA packaging?
nuclear genetic material of eukaryotic exists in two states:
interphase chromosome: cell is not dividing, majority of the time
condensed chromosome: during cell division
What is euchromatin?
loosely packaged DNA that is transcriptionally active, light staining regions
What is heterochromatin?
tightly packages DNA that is inaccessible to transcription, dark staining regions
What are nucleosomes?
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
What are histones?
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
What is higher order packaging?
solenoid or 30 nm filament
coiling of nucleosome filament into higher-order, thicker filament
zig-zag ribbon structure that is wound into a helix
What are non-histone scaffolding proteins?
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
What are mitotic chromosomes?
fully condensed therefore transcriptionally active
What is nuclease?
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
What is an experiment with endonuclease?
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
