The nuclear envelope
-double membrane
-PERINUCLEAR SPACE
-nuclear pores
-outer membrane is continuous with the endoplasmic reticulum
perinuclear space =
the cavity between the two membranes
the eukaryotic cell nucleus
-if the nuclear lamina breaks down, the entire nucleus collapses
-during mitosis lamina induced to breakdown so the nucleus can temporarily fall apart
inner nuclear membrane
inside = filaments = nuclear lamina which give the nucleus structure
nuclear lamina
a NETWORK OF PROTEIN FIBERS that line the inner face of the inner nuclear membrane
-LAMINS belong to a category of cytoskeletal proteins called INTERMEDIATE FILAMENTS
Lamina provides:
1)structural support for the nucleus
2)attachment sites for the chromatin
-therefore it MAY have a role in gene regulation
Things enter and exit the nucleus through the:
nuclear pores
**all are examples of proteins that are transported through the nuclear envelope except:
do: DNA, RNA, polymerase, transcription factors, chromatin assembly proteins (histones), lamins (made outside the nucleus but go inside to function)
DONT: ATP synthase (does enter the nucleus because its functions in chloroplast and mitochondria but never
Nuclear pores
-protein complexes that faciliate transport of molecules through the nuclear envelope
-means of communication between the nucleus and cytoplasm
-not just diffusing through pores (only small molecules can do that) but due to complexity, there is also activ
The nuclear pore complex
-large/ giant complex (50-100 polypeptides)
-complex =120 nm
-opening = 25 nm
Traffic through nuclear pores
-passive diffusion
-active diffusion
-proteins destin to enter the nucleus will have NLS protein
passive diffusion
-small molecules
-small proteins (<50kDa)
-no energy required
active diffusion
-energy-dependent
-required for larger proteins and RNA's
-proteins contain a NLS = nuclear localization signal
nuclear localization signal (NLS)
SV40 T-antigen: initiates viral DNA replication
-the NLS is a 7 amino acid stretch, typically + CHARGED amino acids, in the middle of the polypeptide
-needs to get to the host nucleus so charged
Is a nuclear localization signal sufficient to transport a CYTOPLASMIC protein into the nucleus?
experimental approach:
1) construct a CHIMERIC PROTEIN: pyruvate kinase (protein in cytosol) and NLS from SV40 t-antigen (in an EXPRESSION PLASMID) = recombinant gene
2)introduce the plasmid into cultured cells by transfection
3)observe pyruvate kinase lo
chimeric protein
- had a promoter, pyruate kinase and t-ag NLS
-expression plasmid = because it contains a strong promoter
NLS fluorescence microscopy using anti-pyruvate kinase antibody for detection
A)nuclear localization fo pyruvate kinase with the viral NLS = small distinct white circles
B)cytoplasmic localization of pyruvate kinase with a mutated MLS (the protein fails to translocate to the nucleus) = so its not localized and fluorescences stays i
Protein import through a nuclear pore
the process of transporting large molecules to the nucleus is ENERGY-DEPENDANT
1)protein complex forms
1) protein complex forms
-prospective nuclear protein with an NLS (cargo)
-a NUCLEAR TRANSPORT RECEPTOR protein that recognizes and binds to the NLS in the cargo protein (increases binding to it)
** NLS = part of the cargo protein
Nuclear import
importin interacts with fibrils, moving the cargo into the pores and translocalized into the nucleus. Importin then leaves to pick up another NLS
-this required energy
nuclear transport receptor
recognizes NLS (importin)
Translocation cycle required RNA (GTP- binding protein)
2) entire protein complex (nuclear transport receptor PLUS cargo protein) is translocated through the nuclear pore by sequential interactions with nuclear pore proteins
3)RAN-GTP binds to nuclear transport receptors, causing dissociation and release of th
Ran =
protein that binds nucleotides (SMALL GTP binding proteins 5 kDa) and helps drive translocation of cargo
-can be GTP or GDP bound
Function of Ran in active nuclear import
Ran-GTP is:
-more abundant INSIDE THE NUCLEUS
-it will diffuse to the cytoplasm (out of the nucleus)
-once in the cytoplasm, GTP if hydrolysed to GDP by RAN GTPASE-ACTIVATING PROTEIN (RanGAP), creating a GTP gradient across the nuclear membrane, so Ran-GT
Ran-GDP
-released in cytoplasm diffuses back into nucleus
-the GDP on the Ran protein is then exchanged for a GTP: catalyzed by RAN GUANINE NUCLEOTIDE EXCHANGE FACTOR (RanGEF) in the NUCLEUS
**ability of RAN to exchange GDP to GTP is blocked. what is the most likely effect of this drug on nuclear transport?
nuclear transport receptors would be unable to release their cargo into the nucleus
GTP hydrolysis
-the in the CYTOPLASM
-catalyzed by Ran-GAP (Ran-GTPase activating protein)
-Ran-GDP releases importin, changes conformation + back to nucleus DOWN GDP gradient
GDP/GTP exchange:
-inside the NUCLEUS
-catalyzed by RanGEF (Ran guanine nucleotide exchange factor)
-Ran-GTP binds importin, releases carbon + back to cytplasm DOWN GTP gradient
-replaces GDP to GTP
Nuclear import cycle
-RanGAP= nucleotide exchange factor
-bound to chromatin and only exists in nucleus
-hydrolysis GTP -> GDP causes importin to release energy = allows cycle to restart
-RanGap = bound to cytosolic side of nucleus, in CYTOPLASM
-RanGEF = NUCLEUS
-GDP = high
exportin
bind to cargo in nucleus and makes complex with RanGTP to bring into cytosol (how it leaves)
Regulation of import of NF-kB and Pho4
-NF-kB: transcription factor that exists DORMANT in the cytoplasm in a complex with I kappa B
-Pho4: transcription factor that exists in the cytoplasm in a phosphorylated (inactive) state
NF-kB
-IKB masks the MLS of NF-kB
-if IKB is phosphorylated and destroyed, it releases NF-kB
-NF-kB is then bound by the nuclear transport receptor and translocated to the nucleus, where it can activate transport
Pho4
-the phosphate group blocks its NLS
-pho4 can be dephosphorylated
-upon dephosphorylation, pho4 binds to the nuclear transport receptor and enters the nucleus, where it can activate genes
NF-kB and pho4 cycle:
a signal will initiate the process to remove IKB so the importin can bind and be translocated to active transcription factor in nucleus
Translocation: protein synthesis in the cytoplasm
-translation is protein synthesis by ribosome in the cytoplasm
-translated in cytoplasm but translocated in the nucleus, mitochondria, cytosol or chloroplast
if the synthesis is destined for the nucleus, chloroplast, mitochondrial or stay in cytosol:
translation occurs in a FREE RIBOSOME
if the synthesized protein is destined for the ER and/or golgi as a TRANSMEMBRANE, LYSOSOMAL or SECRETED protien:
translation occurs in the RIBOSOME that are associed with (on) the ROUGH ER and the newly synthesized protein is translocated into the lumen of the ER
-membrane bound = has ribosomes attached to ER proteins with a specific signal sequence that directs it
synthesis of secreted proteins
contain a SIGNAL SEQUENCE at their amino terminus (beginning of the sequence)
-this signal is necessary for translocation into the ER
-after translocation, the signal is removed
signal sequence
8 or more hydrophobic amino acids
(leucine, valine, glycine..)
the signal sequence is SUFFICIENT for directing secreted proteins to the ER
-would never see a protein with ER sequence because its cleaved when entering the ER and would not actually be visible
-if the protein lacks signal sequence it will stay in the cytoplasm
Synthesis and targeting of secreted proteins to the ER
-signal sequences in the growing polypeptide is recognized by the SIGNAL RECOGNITION PARTICLE (SRP)
SRP mechanism
-SRP docks on the SRP receptor on the ER membrane
-the SRPis released, as the growing polypeptide is passed to a TRANSLOCATION CHANNEL
-the translocation channel inserts the polypeptide into the membrane and transfers it across the lipid bilayer
-SIGNAL P
translocation is stalled until:
SRP displayed and is then started
-channel closed until peptide enters the channel
once translocation is completed and the entire chain is in the ER:
the signal peptidase in the ER membrane will signal to stop and the channel will close
-product = polypeptide fully translated in the lumen that can fold into proper conformation in order to transport to the plasma membrane, then outside or somewhere else
synthesis and target of transmembrane protein
-has a hydrophobic stop-transfer sequence which stalls translation because it "gets stuck" and keeps it inside the membrane. It has an alpha-helix which makes it stick to the membrane and keeps in the ER lumen, making it a transmembrane protein
-signal se
Secretion
proteins synthesized on the rough ER take the SECRETION PATHWAY
-endoplasmic reticulum (ER)
-ER-Golgi intermediate complex (ERGIC)
-golgi apparatus
->lysosome (carrying lysosomal proteins)
->plasma membrane ( carrying transmembrane proteins)
->out of the
endoplasmic reticulum (ER)
protein protein processing +synthesizing
ER-Golgi intermediate complex (ERGIC)
protein sorting (initially decides where it will go)
Golgi apparatus
protein sorting (packing and forming transport vesicles to determine where it will go)
secretory pathway
lysosome ->transport vesicles -> fuse with plasma membrane
-some processing in ER and golgi
**a soluble protein that functions within ER lumen is wanted. which is true?
F: signal sequence at c-terminus (cannot be there, must be at end terminus)
F:stop transfer sequence (would be inside the membrane not the lumen)
T: once the signal sequence has been cleaved, the signal peptide will be ejected into the ER membrane and deg
the secretion pathway
note: at the end, the N-terminus translocated to inside of the ER will face EXTRACELLULAR membrane (not in the cytosol) or plasma membrane
endoplasmic reticulum: smooth
-endomembranous system
-smooth ER is the site of LIPID SYNTHESIS AND PROCESSING
-constructs membrane and membrane systems for the cell (steroids, cholesterols = liver)
-contains no ribosomes (no proteins translocated in this part of the ER)
endoplasmic reticulum: rough
-Rough ER is the site or PROTEIN SYNTHESIS AND PROCESSING
-disulfide bond formation, addition of cofactors, addition of oligosaccharides (glycosylation) = made into mature proteins in ER
-its rough appearance is due to associated ribosomes
Golgi apparatus
-GOLGI COMPLEX = the protein sorting center of the cell
-protein-containing vesicles from the ER enter the golgi at the CIS golgi network (enter)
-proteins are then moved through the MEDIAL stacks and released in vesicles from the TRANS golgi (exit)
Golgi function
further oligosaccharide modification of proteins, sorting and packaging either for secretion or delivery to other organelles
golgi secretion pathway
CIS -> TRANS
-when leaving the trans face of the golgi it will either go to the plasma membrane (going outside or to another part of the membrane = transmembrane protein) OR vesicles may go to lysosomes which is a membrane bound vesicle
Transport from ER to golgi
ER-GOLGI INTERMEDIATE COMPLEX (ERGIC)
-system of membrane stacks located between ER and golgi
-sac that looks like the golgi but is between them
Function of ERGIC
checkpoint that ensures that proteins that should stay in the ER (ER resident proteins) do not reach the golgi complex
-retention
reason: traffic from the ER to golgi is NON-SELECTIVE (bulk flow)
**all of the proteins below should be retained by the ER instead of being sent to the golgi except:
STAY = SRP receptor, translocation channel, signal peptidase
SENT TO GOLGI = SRP, hormone receptor
Function of ERGIC: vesicles traffic from the golgi to ER
-ER-resident proteins contain a RETENTION SIGNAL: KDEL (lys-asp-glu-leu) (have signal if they stay in ER)
-if they reach the ERGIC they are recognized, retrieved and sent back to the ER
-they can also be retrieved from the golgi, if htey make it that far
3 types of transport from golgi
1)constitutive
2)regulated
3)transport of lysosomal proteins
1)constitutive
-operates in all cells continuously
-unregulated secretion = bulk flow
-includes secreted proteins, membrane proteins and lipids, extracellular matrix proteins
-occurs at all times
-always cells budding off to go to other parts of hte cell (go to plasma m
2)regulated
-operates in some specialized cells = secretory cells
-these cell types release their secreted products only when stimulated by an EXTERNAL SIGNAL
examples of regulated
-endocrine cells = secrete hormones into bloodstream
-pancreatic cells = secrete exocrine digestive enzymes
-neurons = secrete neurotransmitters
-(only released to outside of cell with external signal but made continuously and stored in vesicles)
secreted products are sorted into:
larger SECRETORY VESICLES that remain in the cytosol until a signal is recieved
signal transduction
signals the vesicle to fuse with the plasma membrane and release cells
**which is true about secretion?
T:the membrane of a secretory vesicle will fuse with the plasma membrane when it discharges its contents to the cells exterior
(budding off is continuous but attaching to the membrane is what is regulated by signals)
Synaptic signaling
-specialized type
-cells signal = electric signal along terminal due to potential
-neurotransmitter not released until it receives its signal
3)transport of lysosomal proteins
-transport of hydrolytic enzymes to the lysosome
-all vesicles that target the lysosome (from the golgi OR plasma membrane as in ENDOCYTOSIS(pinched)) are CLATHRIN-COATED
Clathrin-coated vesicles
-originate from either the golgi or the plasma membrane
-destination =LYSOSOME
-when lysosome proteins contact the inner surface of the golgi, a clathrin coat is assmbeled on the cytosolic side
-the formation of the clathrin coat mechanically induced the
**which about vesicle budding is false?
F: clathrin molecules are important for binding to and selecting for cargos for transport (is not importnat but needs a particular receptor on membrane to choose cargo for transport
T: adaptins interact with clathrin
T:once vesicle budding occurs, clathri
Formation of clathrin-coated vesicles
-lysosomal proteins are recognized by receptors on the inner surface of the golgi
-contact of the lysosomal protein (Cargo)with the receptor induces the assembly of the clathrin coat on cytosolic side
-after the vesicle is released, the coat is shed (unco
Adaptins
capture the receptors and bind clathrin
dynamin proteins
assemble around the neck of the budding vesicle and pinch it off to make a vesicle
selective transport mediated by clathrin-coated vesicles
-transport of cargo across golgi membrane OR plasma membrane
-undergo endocytosis if from extracellular surface
-a similar process mediates the formation of ENDOCYTIC VESICLES on cytosolic surface of the plasma membrane
naked transport vesicles
direct to lysosome
the lysosome
-DIGESTIVE ORGANELLE
-molecules taken up by ENDOCYTOSIS (or phagocytosis)
-obsolete or damaged components of the cell may also be taken to the lysosome by AUTOPHAGY
- the breakdown enzyme is contained in one compartment not all over the cell
autophagy
damaged components of cell are broken down covering itself with another membrane os it doesnt get toxic in the cell
lysosome contains ACID HYDROLASES:
-these enzymes need a highly acidic environment to function
-environment inside the lysosome is more acidic (ph=5) than the cytoplasm (ph=7)
-more acidic inside lysosome than outside due to proton pump
** if a lysosome breaks, what protects the rest of the cell rom lysosomal enzymes?
cytosolic pH
lysosome internal pH
-the digestion of materials by the cell requires the formation of mature lysosomes
maturation of lysosomes
-EARLY endosome = forms from the fusion of endocytic, phagocytic, or autophagocytic vesicles (has proton pump so is slightly acidic)
-LATE endosome = forms when hydrolyase-containing vesicles from the golgi fuse with early endosome
-late endosome further
lysosome is only functional when it is mature = ph?
ph=5
-only called a lysosome when ph is 5 since that is when acid hydrolyase can function
phagocytosis:ingestion of other cells or particles
1)long = neutrophil ingesting a dividing bacterium
2)circular = macrophage ingesting damaged red blood cells (PSEUDOPODS = extensions of the membrane of phagocytic cells that extend over the ingested cells (indicated by arrows))
Receptor-mediated endocytosis
specific molecules or particles are recognized by cell surface receptors and taken up by endocytosis
ex of receptor-mediated endocytosis = endocytosis of cholesterol
-cholesterol is extremely water-insoluable (must transport in protein complexes LDL)
-it is transported from the liver to the bloodstream and around the body bound to protein in the form of particles: LDL
-efficient way to take material from extracellular
LDL
-low density lipoproteins ("bad cholesterol"
-recognized by surface LDL receptors, taken up by the cell and delivered to the lysosome
-is dissociated by digestive enzymes in the lysosome and cholesterol is released for membrane synthesis
-LDL receptors ar
hydrolytic enzymes
break down LDL and release cholesterol in cytosol (for smooth ER that needs it)