Cell Bio and Genetics Exam 3 Flashcards
components of the endomembrane system
ER, Golgi, Vesicle, Lysosomes and peroxisomes
What does the endomembrane system do?
compartmentalization of cell metabolism
proper trafficking of proteins and lipids to their appropriate destination
how do proteins get where they are going?
a signal sequence determines whether they will assume a cytosolic or secretory pathway. A signal sequence is a recognizable. sequence that directs protein as it is being translated to the ER(This is what makes the ER rough)
what is the ER? what is the smooth ER
A network of lipid bilayer in the cell. The smooth ER sequesters calcium (important for cell signaling), involved in the synthesis of steroid hormones, synthesizes enzymes for detoxification, and enzymes for glucose release.
what is the rough ER (RER)
full of ribosomes are bound to it owing to the translation of protein with a signal sequence. The RER is the site of synthesis of membrane-bound, secretory and lysosomal proteins
ER lumen
an internal compartment that never “sees” or comes in contact with the cytosol
evidence for localization of secretory proteins to the ER compartment experiment 1
One can mash up cells to yield “microsomes”. First proteins are radio labeled and a protease is added. The proteins will remain intact or add detergent and then protease. In this instance, the radiolabled proteins will be degraded by protease.If proteins were in the cytosol of the cell, they’d be chewed up, but because they are in the lumen of the RER, they are protected from proteolysis. When detergent is added proteins are released from microsome and become susceptable to protease activity. Conclusion: proteins are located inside of the RER lumen
evidence for cotranslational translocation of secretory proteins
translocation of a protein is coupled to its translation. That is, proteins are moved into the ER lumen as they are being synthesized.
Ribosomes with associated proteins that carry a signal sequence. add microsomes incubate and find that proteins don’t end up inside of the microsomes. However if microsomes are added earlier while the proteins are still being synthesized then the proteins end up inside the microsomes. So proteins move into the ER as they are being made.
Signal recognition protein (SRP)
signal sequences have a high affinity for SRP, hence binding occurs. When SRP binds translation stops. binding causes a conformational change in the translocon from a closed to an open form so the polypeptide can start fitting into it. SRP-SRP receptor complex is responsible for guiding the protein into the RER translocon. GTP strengthens the interaction of SRPs with SRP receptors.
translocon
a structure or gate through which proteins move
How are SRP-SRP receptors released from the complex?
hydrolysis of GTP into GDP and SRP is recycled back to its guiding jobs. The signal sequence is left in the translocon
How does translation continue after the protein moves into the translocon?
The protein moves through the translocon into the ER lumen. Proteins fold and will then move into the Golgi and into secretory vesicles, and out of the cell.
What mechanisms support protein folding?
chaperone proteins hep other proteins fold if they can’t fold spontaneously. This is why translation haooens before the protein is translocated. For a protein to be secreted all of these processes must occur during protein synthesis.
proteins are directed to the er if…
they are destined for the er, Golgi, lysosomes, secretion, or the plasma membrane
proteins are directed to the cytoplasm if..
they are destined for the nucleus, mitochondria, peroxisomes, or the cytoplasm
The smooth ER does…
synthesizes lipids(cell membrane, steroids), metabolizes carbs, aids in detox of drugs
the rough ER does…
protein synthesis and post translational modifications(disulfide bridges)
the Golgi apparatus does..
modifies proteins made in rer, sorts and sends proteins to proper places, and synthesizes molecules for secretion
parts of the Golgi and where they are
cis stack, closest to the er, medial stack, between the cis and trans, trans stack closest to the cellular membrane
cisternea
membrane folds in the endoplasmic reticulum
How does transport from the ER to the Golgi
vesicles for secretory proteins and integral proteins
How are proteins oriented after transport
secreted proteins in the ER lumen will become oriented to the outside of the cell after transport. what’s oriented on the systolic side of the ER will stay in the cytosol after transport.
Nearly all proteins that are destined for the cell membrane undergo…
glycosylation. there are two ways n-linked vs o-linked(refers specifically to the linkage that occurs between sugars and an amino acid)
What is the difference between n-linked and o-linked
all n-linked glycosylation occurs in the ER, and the N-linked recognition sequence includes an aspargines (N) in the middle. O-linked sugars are added a serine residue, a process that occurs primarily in the Golgi.
The organization of the endomembrane system starting at the nucleus
nucleus, rough ER, smooth ER, cis Golgi network, medial Golgi, trans Golgi network, vesicles, cytoplasmic membrane
When can secretion be regulated
once a protein is vesicularized an example of such secretion occurs in nerve cells
How do enzymes get into lysosomes?
via fusion of vesicles with the lysosome
What is the mechanism that moves things within the Golgi?
cisternal maturation and vesicle movement. Vesicle transport mediates the movement of protein from the ER to the Golgi, and then from the Golgi to vesicles, In the Golgi itself both cisternal maturation and vesicle transport is likely to occur
What is the problem with cisternal maturation
different enzymes/proteins are present in the cis Golgi than are present in the medial and trans Golgi. if movement through the cistern was a matter of cisternal maturation, all proteins within all compartments of the Golgi should be the same
What is the problem with vesicular movement
vesicle transport is not unidirectional. Rather you can have retrograde movement
How does one study membrane trafficking through biochemical approach
lyse cells and perform sub cellular fractionation of membrane study the membrane components. After fractionation, isolate proteins from membrane fractions and run then on a gel. In this way, proteins that are associated with a particular fraction may be identified.
How does one study membrane trafficking through genetic approach
make mutants and see where the protein accumulates. For instance, Class A mutants may result in the accumulation of proteins in the cytosol, when in fact the proteins are supposed to be secreted! such a defect may reside in transport to the er. Class B mutants cause proteins to accumulate in the RER.
vesicles are coated with proteins, what are the types of vesicle protein coats?
COP1, COP2 or clathrin. coated vesicles form off the ER or Golgi
Cathrin coated vesicles….
carry proteins from the trans Golgi network to endoscopes/lysosomes
COP 2 coated vesicles…
carry proteins from the ER to the Golgi
COP 1 coated vesicles…
plays a role in retrograde transport of proteins from Golgi back to the ER
How do coated vesicles form?
SAR- a protein that binds to GTP and acts as a G protein. SAR can recruit other COP2 proteins
there are proteins resident in the ER too, but proteins that need to move onward….
are put into coated vesicles via cargo protein receptors that have a specific binding site for proteins with a certain motif
What happens when proteins get misdirected
they get moved back to where they’re supposed to be via retrograde transport
What protein is involved in protein stripping
SAR because it has GTPase activity. GTP is hydrolosized to form GDP that causes a conformational change in the proteins that constitute the coat so the coat falls away
how do mis-sorted proteins get from the Golgi back to the ER?
cis Golgi network is a KDEL peptide that can bind to KDEL binding sites that are present on proteins. In addition, the KDEL receptor has a sequence on its cytosolic tail that binds specifically to COP1 coat proteins
how do things get into the lysosomes?
from the ER-cis Golgi- trans Golgi. these proteins have a particular recognition sequence that recruits mannose 6 phosphate. Clathrin vesicles have a mannose-6-phosphate receptor that interacts with mannose-6-phosphate. only proteins with mannose-6-phosphate represent those that are supposed to go into lysosomes.
two examples of retrograde transport
- if a protein is supposed to be in the ER but has been mis-sorted to the Golgi, a KDEL sequence on an ER luminal protein interacts with a KDEL receptor, and a sequence on an ER membrane protein interacts with COP1
- mannose-6-phospohorolation occurs on all lysosomal proteins. manse-6-p is recognized by mannose-6-p receptors present with clathrin-coated vesicles.
neuronal vesicle model
post-synaptic vesicles sit and wait for a cell signal before fusing with the target membrane. The action of neuronal v-SNAREs and t-SNAREs is involved in the interaction between vesicle membranes(where v-SNAREs are located) and target membranes(where t-SNAREs are located)
Synaptobrevin(VAMP) is…
a v-SNARE. when neuronal cell recieves a calcium signal(Ca+ influx), three proteins become intertwined via alpha helices to lock membranes together. These three proteins are VAMP, SNAP-25(a t-SNARE) and syntaxin(another t-SNARE).
vesicle docking
involves Rab. Rab-GTP binds to a Rab effector, but only after Sar-GTP is cleaved by its GTPase to remove the proteinaceous coat associated with the vesicle. Once bonded to the Rab effector, the SNARE complex can form, wrap together, and intertwine. This allows membranes to fuse.
NSF
a general factor involved in these membrane fusion events, uses energy from ATP to allow release of SNARE complex
What is the importance of vesicle docking?
Mutations that disrupt vesicle docking are lethal
What is the importance of different Rab proteins and SNAREs?
this is what provides specificity so that certain vesicles are targeted to certain membranes. Rabs are involved in initial recognition. SNAREs are involved in recognition and membrane fusion
Overview of vesicle docking
Rabs bind via tethering proteins=the first recognition event
v-SNAREs bind to certain t-SNAREs to form a SNARE complex
Once bound, proteins wrap together to allow membrane fusion
To pull the complex apart, NSF, via and energy-dependent mechanism(ATP-ADP) drives the SNARE complex to unravel and fall off
Medical relevance of vesicle docking
Clostridium botulinum and Clostridium retain are both toxin producers, these toxins cleave SNAREs. Then no membrane fusion can occur and there is no release of neurotransmitters causing paralysis.
Medical relevance of vesicle docking
Clostridium botulinum and Clostridium retain are both toxin producers, these toxins cleave SNAREs. Then no membrane fusion can occur and there is no release of neurotransmitters causing paralysis.
Polycystic kidney disease
renal cyst formation equated with renal failure. cyst formation is the result of messed up syntaxin 3 and 4. This inappropriate expression of syntax’s traffics proteins to the wrong places. cell architecture is messed up and hence abnormal tissue organization occurs. an example of vesicle docking disruption.
Bulk-phase
nonspecific uptake if extracellular fluid. all cells do this and it is not triggered by anything imparticular
receptor-mediated
specific uptake of extracellular molecules via receptor-ligand interactions. not all receptor-ligand interactions involve endocytosis; that is, not all receptor-ligand interactions result in ligand incorporation into the cell
the process of receptor-mediated endocytosis
first, the ligand binds to the receptor. Next, clathrin and adaptor proteins bind to the receptor to form an early coated pit. Then, a fully coated pit is formed as the process pf endocytosis takes up the ligand into the cell. Once the pit pinches off from the plasma membrane (while it is associated with clathrin coat) is called an endosome. The clathrin coating is then lost to yield a transport vesicle.
How does the clathrin coat form?
clathrin becomes organized as a triskelion, individual triskelion come together to form a geodesic cage
How do endoscopes age?
move deeper into cells they “age”. early vs late endosomes differ both positionally(present deeper within cells) as well as physically.
within endosomes there are proton pumps. What function of they serve
protons are pumped into the interior (or lumen) or the endosome. early endosomes have a certain pH. relatively speaking, late endosomes have had more time to pump protons into them and have a more acidic pH.
receptor recycling
once the ligand has been taken up. that is, only the receptor gets cycled back to the cell membrane to bind another receptor
examples of receptor-mediated endocytosis
LDL in the blood binds to receptors. That is, an LDL particle (which is a protein to which cholesterol is bound) binds to LDL receptors that are associated with clathrin-coated pits. coated vesicles form next, then an early endosome when the vesicle pinches off from the plasma membrane. The clathrin coat is lost and a late endosome ph 5 develops[LDL receptor gets recylced]. At pH 5 cholesterol is released from LDL so it is released into the cell cytoplasm. The end result is that cholesterol has been removed from the blood and moved into cells. Intracellular cholesterol functions as a signal to tell the cell whether more cholesterol is needed for synthesis or not.
components of ECM
collagens, fibronectins, laminins, proteoglycans
functions of ECM
gastrulation(invagination and mesenchyme ingression), keratinocytes moving on collagen, neural crest cell movement
cell adhesion molecules
molecules at cell surface that interact with ECM components
1. intigrins
2. selectins
3. IgSF CAMs
4. Cadherins
collagen
fibrillar- types 1,2,3
non-fibrillar- type 4(found exclusively in basement membranes/form networks)
lots of collagen in corneas laid down in patterns perpendicular to one another to add strength to the tissue.
fibronectins
heterodimeric proteins/ two polypeptides connected by S-S bonds
involved in interconnections between ECM components and direct cell migration
Structure of fibronectins
linear scheme, modular having functional domains and the 2 polypeptides are connected via cysteine residues
comprised of three different regions and different binding affinities and functions
type 1 region binds proteoglycan and fibrin(heparin sulfate is part of proteoglycan)
type 2 region binds collagen
type 3 region binds integrins(arginine, glycine, aspartic acids
laminins
can effect cell migration, growth and differentiation
laminins are heterotrimers
they take. on a whirly type of conformation
different binding domains: one that binds sulfate lipids, another that binds collagen and sulfate lipids, and another binds integrins
proteoglycans
made up of chains of glycosaminoglycan(GAG)
examples: chondroitin sulfate, keratin sulfate, heparin sulfate and hyaluronic acid
made by linking a glycosaminoglycan chain to a core protein
ECM in cell movement how the mesoderm is formed
As mesoderm is formed, cells migrate into the center of the gastrula. This cell migration into the center of the gastrula is guided by components of the ECM
ECM in cell movement (how the gut is formed)
Cells invaginate using proteoglycans
keratinocytes moving on a collagen coated surface
Other round cells in the field do not move, because they are mutant cells missing a particular integrin
neural crest movement
neural crest cells move along fibronectin as the neural fold becomes a neural tube. fibronectin guides the movement of neural crest cells move outward from a neural fold on a slide coated with fibronectin
Integrins
mediate connections between cells as well as between cells and the ECM, composed of two different chain types(alpha and beta) which undergo conformational changes to assume active and inactive formations. lipids bind to beta chains.
What contributes to variation in integrins
there are different types of alpha chains and beta chains that can be mixed and matched and both subunits are used to bind ligand
selectins
transmembrane proteins with important domains. EGF domains are for epidermal growth factor. Lectin domains bind to carbs and determines specificity and recognizes carbohydrates . have variable numbers of cystine-rich domains.
IgSF CAMs
all posses a number of Ig domains, and. hence they comprise part of the Ig superfamily. Ig domains are recognition domains.
cadherins
cadherin domains, zipper-like binding, Ca2+ dependence hemophilic binding
Characteristics of cell adhesion molecule binding
Ca2+ dependence or independence
hemophiliac vs heterophilic interactions (cadherins are almost always hemophilic, integrins are always heterophilic, selectins are always heterophilic, IgSF CAMs are almost always hemophilic
types of cell junctions
desmosomes and adheres junctions- provide tissue integrity and strength, connection to the cytoskeleton, intercellular communication
gao junctions(connexons)- communication between cells, coordination of tissue response
tight junctions
why do the mesodermal cells migrate to the inside and ectodermal cells to the outside
this is because of the differences in the affinities of mesodermal cells for one another and ectodermal cells for one another. In fact, the mesodermal-mesodermal affinity is stronger, and so it homes to the center as the weaker ectodermal-ecrodermal cells get pushed to the outside of the embryo. Can also preform this experiment with cells that harbor cahdrens and cells that harbor IgSD-CAMs. Homophilic interactions will give rise to two distinct layers
roles of cell adhesion molecules
tissue organization and integrity
communicatio b/T cells
proper migration and homing
why are hemophiliac reactions important
these interactions are important to the making of tissue
experiment to prove hemophiliac interactions are important
take an embryo and add proteases to separate ectodermal and mesodermal cells into single cell suspensions. Then, mix the single cell suspensions together and watch! The cells organize themselves into ectodermal tissue and mesodermal tissue.
why do the mesodermal cells migrate to the inside and ectodermal cells to the outside
this is because of differences in the affinities of mesodermal cells fro one another, and ectodermal cells for one another. In fact, the mesodermal-mesodermal affinity is stronger, and so it homes to the center and the weaker ectodermal-ectodermal cells get pushed to the outside of the embryo
Roles of cell adhesion molecules
tissue organization and integrity
communication between cells
proper migration and homing
tissue organization and integrity
desmosomes- give integrity to cell layers
adherens junctions- give integrity to cell layers
hemidesmosomes are like desmosomes except they do not connect cells with other cells; rather they connect cells to components of ECM
how about intercellular communication
gap junctions are tunnels in the membrane that allow neighboring cells to exchange things. The gaps of “gap junctions” are bigger than channels to allow signaling molecules to pass from cell to cel
tight junctions
rather tight junctions prevent the flow of molecules in the intercellular space
two different types of chromatin
the electron dense chromatin is concentrated along the periphery of the nucleus, heterochromatin. the electron non-dense region is localized centrally to the nucleus and is called euchromatin
what is heterochromatin
Dan that is more compact and transcriptionally inactive
what is euchromatin
Dan that assumes a more open conformation making it transcriptionally active
during interphase what chromatin predominates?
euchromatin
What is facultative heterochromatin
transiently compact, only compact during phases of an organisms life ie. Barr bodies in females and calico cats
What sequence causes transcription to stop on the RNA
AAUAAA, this is called a polyadenylation sequence
What is the core promoter sequence and where is it
-25 and TATAA and the core promoter is always at the same spot relative to the start of the sequence.
How does one show that a particular sequence is important?
by preforming deletion analysis within promoter region of the gene and replace the structural gene with a reporter gene. If green fluorescence results transcription is on. activity derived from the core promoter alone gives basal level transcription
transcription factors
proteins that bind to DNA elements to promote/regulate basal level transcription
1. the basal transcriptional machinery includes the promoter and operator.
2. transcriptional activators and repressors operate at enhancer and silencer sequence.
basal transcriptional machinery
the TATAA box is the site at which an entire complex of proteins form a “preintiation complex”. transcripts have a start(initiation), middle (elongation), and end(termination).
transcription factors are designated for
transcription factor for RNA pol 2, and then a letter designated follows to distinguish one from another.
how do transcription factors come together? -30 complex formation
the TATAA box becomes bound by a huge multi-subunit factor called TFIID. TFIID includes a TATAA binding protein, TBP, and other proteins that are associated with it called TAFs. Once TFIID binds to TATAA it recruits TFIIA and TFIIB which stabilize the protein complex. Then RNA pol 2 is recruited and has its own TFIIF. complex comes together at -30 on DNA so that RNA pol 2 is positioned at -25 where it needs to be to begin gene transcription. TFIIE comes and TFIIH(a kinase) which phosphorylates the carboy terminus of RNA pol 2 which activates transcription.
chromatin
eukaryotic chromsomal DNA and associated proteins, part of transcriptional control
histones
small, conserved, positively-charged proteins with lysines and arginines that assume ionic interactions w DNA. High salt concentrations can disrupt these interactions
nucleosomes
bead like units comprised of DNA and associated histones H2A, H2B, H3 and H4
core particle
an octamer of H2A, H2B, H3 and H4 histones about which 200bp of DNA is wrapped. H1 is associated with the linker DNA between nucleosomes
Chromatin re-modeling enzymes
- ATP-dependent re-modeling enzymes (Sin/NurD)
- histone acetyltransferases(HATs) and histone deacetylases (HDACs)
HATs are typically…
HDACs are typically…
transcriptional activators
transcriptional silencers
