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Ch 4 Histology Objectives
Terms in this set (56)
Describe how proteins are targeted to their final destination by the presence or absences of targeting sequences including proteins bound for the "Cytosol"
•Proteins bound for the cytosol are made on free ribosomes.
•Once protein synthesis on a free ribosome is complete, the protein is discharged to the cytosol.
•If the protein does not contain any specific targeting sequence, it will have no place to go, so it will stay in the cytosol.
Describe how proteins are targeted to their final destination by the presence or absence of the targeting sequences to the "Nucleus"
For the nucleus, proteins contain a sequence of amino acids as part of their structure called the nuclear localization sequence (NLS).
-- In order for the proteins to get into the nucleus (where they belong), they must travel the nuclear pore complex, which regulates what can and cannot enter the nucleus.
While targeting proteins to get into the nucleus, the NLS is used to get in the nucleus how ?
The Nuclear Localization Sequence (NLS) of these proteins binds to another protein called importin. Importin is allowed to traverse the nuclear pore complex, bringing with it the protein of interest.
Once the protein of interest, is inside the nucleus what takes place? And what other protein binds to cytosol?
Importin is released and exported back to the cytosol for recycling.
Ran, is the protein when bound to GTP (RanGTP) , it is able to bind to importin and causes dissociation of importin- cargo complex.
After Importin-RanGTP returns to cytosol through nuclear pore complex, in the cytosol what does RanGTP cleave?
In the cytosol...
•Ran (has GTPase activity) cleaves GTP à GDP (RanGDP)
•RanGDP dissociates the importin.
What is the requirement for any protein that leaves the nucleus? And What is this process called?
Any protein that is required to leave the nucleus must contain a sequence of amino acids identifying that protein as having permission to leave , this sequence is called the nuclear export sequence (NES)
How does a protein with NES leave the nucleus?
•A protein in the nucleus containing a NES will bind to another protein called exportin, along with RanGTP.
•The cargo-exportin-RanGTP complex then moves through the nuclear pore complex to the cytosol.
•The GTPase activity of Ran will then cleave the RanGTP to RanGDP, which causes the cargo to undock from this complex.
How are mRNA and tRNAs able to leave the nucleus given that they are not proteins and therefore cannot have an NES?
•Remember that mRNAs and tRNAs leave the nucleus bound to a series of proteins (TAP, PABP, translation factors, etc.).
•TAP, which contains a NES, most likely allows the mRNA to bind to exportin and leave the nucleus.
•In the case of tRNAs, there is a specific exportin (exportin-t) that binds to mature tRNAs and allows them to leave.
•A protein with a NES is not required for the export of tRNA from the nucleus.
The overall process of the cargo with NLS is ???
In the cytosol, importin binds to the cargo destined for the nucleus (which contain NLS), importin then shuttles the cargo across the nuclear pore complex, and then Ran unloads the cargo from importin and brings importin back to the cytosol.
Describe how proteins are targeted to mitochondria, requiring mitochondria sequence...
First we need to know , Mitochondria contain their own DNA, and can make their own proteins as well. The majority of mitochondrial proteins are made on free ribosomes; it must contain a mitochondrial targeting sequence to get to the mitochondria.
List the 4 compartments in the mitochondria that proteins target?
•outer mitochondrial membrane (OMM)
•inner mitochondrial membrane (IMM)
In order to target a mitochondrial protein to its correct destination, the mitochondrial targeting sequence needs to be able to specifically associate with mitochondrial transporters. What are the transporters?
•TOM: translocase of outer membrane
•SAM: sorting and assembly machinery (OMM)
•MIA: mitochondrial intermembrane space import and assembly machinery (IMS)
•TIM-22: translocase of inner membrane-22
•TIM-23: translocase of inner membrane-23
For any protein that is required to go to the mitochondria MUST be able to bind to TOM, which allows the protein through the OMM. What needs to be done to ensure that the protein does not fold prior to entry through TOM ( which would impede the protein getting through)?
A heat- shock protein 70 (hsp 70), a chaperone protein, binds to the mitochondrial protein to prevent premature folding.
Once a protein docks on TOM and gets into the mitochondria, it can associate with one of the other mitochondrial proteins., and List those mitochondria proteins.....
•SAM, MIA, TIM-22, or TIM-23
•Association is based on its target sequence
Once the protein has reached its final destination within the mitochondrial, what takes place?
The mitochondrial targeting sequence may be removed by enzymes known as mitochondrial processing peptidases.
Describe how proteins are targeted to Peroxisomes
All peroxisomal proteins are encoded in the nuclear genome (unlike mitochondria), and are synthesized on free ribosomes.
There are 2 Peroxisomal targeting sequences called PTS known as PTS 1 and PTS 2.
PTS 1-- it is a sequence of amino acids found on the C-terminus of the protein.
PTS 2-- sequence of amino acids found on the N- terminus of the protein.
What are the mechanism for targeting proteins to the "Perioxsome"
The Peroxisomal targeting sequences (PTS) will be bound by a PTS receptor, known as peroxin, in the cytosol.
PTS 1---> receptor is Pex 5
PTS 2---> receptor is Pex 7
Once the peroxin is bound to the protein, it can dock onto the peroxisome importomer.
Explain the peroxisome importomer....
It is found on the peroxisomal memebrane , that is responsible for transport.
It will translocate the peroxin/PTS- containing protein into the matrix of the peroxisome.
Peroxin can then be returned to the cytosol via translocation through the importomer for recycle.
The difference in proteins targeted to mitochondria vs the perioxisomal is....
The importomer can translocate proteins that have already folded; no chaperone proteins are required to bind to the peroxisomal protein to prevent binding.
Explain Zellweger Syndrome including incidence , basic genetics/ mutation...
Zellweger Syndrome is the most common disorder of peroxisomes. The highest incidence of the disease occurs in Quebec, Canda.
-- It is a autosomal recessive disorder caused by mutation in the genes that encode for peroxins.
What are Peroxisomes involved in ?
•metabolizing very long-chain and branched-chain fatty acids
•metabolizing hydrogen peroxide
•Patients with Zellweger Syndrome nervous system phospholipids become highly concentrated.
What is the presentation of Zellweger syndrome?
These patients typically present with, complications of the central nervous system:
-- Progressive blindness
-- Very poor muscle tone and coordination
-- enlargement of the liver, and cysts in the kidney
•There is currently no universally accepted treatment and children born with it typically do not survive longer than one year.
What are the CNS effects , liver enlargement, and cysts in the kidney due to:
The CNS effects are due to the inability neurons to properly myelinate as the synthesis of plasmalogens is impaired.
The liver enlargement and kidney cysts are due to the accumulation of very long-chain fatty acids that cannot be metabolized.
*Remember concerning the bound ribosome......
Once a protein has been synthesized on a bound ribosome, it is now in the rER. And are then sent to the Golgi to be further processed before being sent to their final destination.
Describe basic appearance and structure of the Golgi...
Appears as a flattened stack of membranes similar to the rER; without ribosomes, and not continuous with the nuclear membrane and ER.
What is the Golgi primarily involved in ?
-- Post- translational modifications ,sorting, and shipping proteins recently synthesized by the rER,
-- the number of Golgi bodies
-- the size of these Golgi bodies in any particular cell vary with the amount of protein produced by that cell.
-- The Golgi is so large that it can be visualized on a light microscope.
Compare and contrast the location, function, and composition of the cis- and trans- Golgi.
•The two faces of the Golgi are not identical.
•primarily responsible for receiving cargo from rER
•faces plasma membrane
•involved primarily in shipping cargo to other destinations
•primarily involved with post-translational modifications of proteins as they move through the Golgi.
What are the difference in protein content in the different Golgi regions?
cis-Golgi contains a higher concentration of coatomer proteins
•Coatomers are involved in shipping material back and forth between the rER and Golgi
trans-Golgi has a higher concentration of clathrin
•Clathrin is another coatomer protein
•involved in shipping material out of the Golgi
Media Golgi region contains many enzymes involved in post-translational modification of proteins moving through the Golgi.
The membrane becomes thicker moving from the cis- to the medial- to the trans-Golgi regions.
Why does the membrane becomes thicker moving from the cis- to the medial-to the trans- Golgi regions?
The changes in the thickness of the membrane are primarily due to the cholesterol content of the membrane, which causes thickening of the membrane.
Explain the path of COP- II coated vesicles from the rER to the Golgi.....
COP-II coated vesicles
•transport proteins recently made in the rER to the Golgi
•vesicles are produced by pinching off pieces of the rER using a coatomer protein called COP-II (coat protein complex-II)
•vesicles are accepted by the cis-Golgi network
This type of transport is known as anterograde transport, moving from the center of the cell towards the periphery of the cell.
Explain the path of COP- I coated vesicles from the Golgi to the rER.
•If a protein is ultimately destined for the ER, it will need to be transported via retrograde transport back to the ER from the Golgi.
•ER retention signal
•sequence of amino acids near the C-terminus
•found on ER-resident proteins
•2 well-described ER retention signals:
•KKXX/KXK (lysine-lysine-X-X or lysine-X-lysine)
•X is any amino acid
In order for the Golgi to send proteins back to the ER , what must we do?
Identify those proteins belonging to the ER, which are the ER resident proteins .
They contain sequence of amino acids near the C-terminus on them which are called the ER retention signal.
Compare and contrast anterograde and retrograde transport and apply to vesicle transport between the rER and Golgi.
When retrograde transport takes place--- , proteins that were destined to go to the ER now need to be transported back to the ER from the Golgi.
Anterograde transport is moving from the cneter of the cell towards the periphery of the cell. So basically saying the proteins recently synthesized in the rER are sent to the Golgi in COP- II coated vesicles. These vesicles are accepted by cis Golgi network...
What the 2 well described ER retention signals and explain how they are used to bring ER resident proteins from the Golgi to the rER?
2 well-described ER retention signals:
•KKXX/KXK (lysine-lysine-X-X or lysine-X-lysine)
•X is any amino acid.
Some proteins that were made on bound ribosomes and incorporated into the rER membrane were KDEL and KKXX/KXK ER retention signal receptors.
•When the Golgi has received this cargo, the ER retention signals bind to the ER retention signal receptors.
•The Golgi then pinches off pieces of the CGN membrane containing the ER retention signal receptors using COP-I and sends those vesicles back to the ER.
•Proteins that are destined to stay in the Golgi are not shipped out only to be shipped back. Instead, the Golgi maintains these proteins.
Explain how Golgi kin recognition and the transmembrne-domain mediated retention are used to maintain Golgi- resident proteins in the Golgi?
Golgi kin recognition
•Golgi-resident proteins form polymers with each other due to C-terminus interactions
•Polymers are too large to be shipped out of the Golgi and are maintained
•Golgi-resident proteins also use the mechanism of transmembrane-domain mediated retention to stay in the Golgi.
•TransMembrane-bound Golgi-resident proteins move through the Golgi in anterograde fashion until the membrane thickness matches the thickness of the hydrophobic transmembrane-spanning region
•Once the two thicknesses match, the protein is essentially trapped and cannot move further forward.
Proteins that are not intended to stay in the Golgi will eventually make their way to the Trans - Golgi Network (TGN). Where does the TGN place them
The TGN will place these proteins into clathrin-coated vesicles to be sent forward.
Explain how proteins destined for the plasma membrane are packaged and shipped from the trans- Golgi network (TGN) and the fate of the proteins once these vesicles fuse with the plasma membrane.
•Once the clathrin-coated vesicles leave the TGN, their default destination is the plasma membrane.
•When these vesicles fuse with the plasma membrane, proteins that were embedded within the vesicle membrane become incorporated into the plasma membrane itself.
•Proteins that were dissolved in the lumen of the vesicle will be released to the outside of the cell.
Compare and contrast proteins transported from the Golgi by clathrin-coated vesicles and secretory vesicles including the pathways of each and association with the plasma membrane?
In the clathrin-coated vesicle--- the pathway from the rER to the Golgi to the plasma membrane/ extracellular space is unregulated in the sense that once the vesicle leaves the Golgi, it is automatically sent to its final destination and cannot be stopped or interrrupted, which is called the constitutive secretory pathway.(unregulated) it leaves TGN and goes straight to plasma membrane.
Secretory vesicles - the only difference is their fusion with the plasma membrane is not automatic!! Its regulated---- This pathway is called the regulated secretory pathway. so basic ---•Vesicles containing crystallized proteins are released from the TGN in vesicles than can only associate with the plasma membrane under certain conditions.
•Over time, the cell will fill with these secretory vesicles.
•The vesicles will fuse with the plasma membrane when conditions are favorable, releasing their contents into the extracellular fluid.
•This is how most glandular cells release their hormones and how neurons release their neurotransmitters.ally this is the association with the plasma memebrane:
How proteins for the secretory pathway may become crystallized in the Golgi.?
As the proteins move through the Golgi , they ecome associated with ions, particularly calcium and zinc. Because of the association with ions, as well as changes in the pH of the Golgi cisternae, the proteins crystallize.
List and describe each stage of insulin synthesis for Pre-Proinsulin
- signal peptide which directs the ribosome to the rER
- signal peptidase cleaves the signal peptide- This is done upon completion of preproinsulin synthesis, then placed in the lumen of the rER. Which results in product proinsulin.
List and describe the stages of insulin synthesis for Proinsulin
-in the rER Proinsulin is folded into its correct confromation and three disulfide bonds are formed.
- Proinsulin is placed in COP-II coated vesicle and transported to the CGN.
-As it travels through the Golgi, a series of peptidases cleave proinsulin into insulin by cutting a chunk out of the C-peptide protein.
List and describe the stages of Insulin
-Insulin is still in one piece as there are disulfide bonds that hold the N-terminus (B-peptide) and C-terminus region (A- Peptide) together.
-Insulin molecules form a hexamer due to interaction with Zinc, which crystalizes the insulin.
- Due to crystallization the Golgi sends insulin into secretory vesicles instead of directly transporting them to the plasma membrane..
Explain where insulin is stored and the condition causin it to be released from secretory vesicles.
Insulin is stored in the pancreatic endocrine cells until changes in these cells causes the vesicles to fuse with the plasma membrane..
- which causes "INCREASE" in glucose entering the cell., leading to the intracellular concentration of calcium to increase.
- Calcium is what causes the secretory vesicles to fuse with the plasma membrane, releasing the insulin crystals and C-peptide.
Describe the active form of insulin...
The plasma concentration of zinc is very low, the zinc that crystalized the insulin dissolves into the plasma, causing the insulin hexamers to become insulin monomers, which is the active form.
Describe the C- peptide and explain why it is and important clinical marker......
C-peptide the marker of endogenous insulin production, which helps to determine whether you're measuring endogenous insulin or insulin that is given to self.
FYI: C-peptide measures the insulin production in a patient.
What are the 2 sequences necessary to designate a protein for the endosomal/lysosomal pathway?
1) These proteins have already received a core oligosaccharide on an asparagine residue.
2) Another sequence is necessary to designate a protein for this pathway; this sequence is called the signal patch, and it is quite variable.
List the steps of M6P post translational modification and vesicle packing for endosomal/lysosomal pathway proteins already having a core oligosaccharide and a signal patch.
•Proteins with a core oligosaccharide and a signal patch will encounter a phosphotransferase in the Golgi
•phosphorylates one of the terminal mannose residues on the core oligosaccharide, forming a mannose-6-phosphate (M6P) residue.
•Proteins destined for endo-lys pathway will become clustered together in the TGN
•due to the presence of an M6P receptor (M6P-R)
•M6P-R is a membrane-bound protein that also came from the rER
•The M6P residues bind to the M6P-Rs, then Golgi pinches the vesicles off.
List examples of endosomal/lysosomal pathway proteins and classify as acid hydrolases, hydrogen pumps, or highly glycosylated proteins.
•Acid hydrolases (most): enzymes that break down lipids, proteins, carbohydrates, and nucleic acids and are only active at an acidic pH)
•Hydrogen pumps: ATP-dependent proteins that acidify the contents of the lysosome
•Highly glycosylated proteins: associated with the lysosomal membrane to prevent autodigestion
•lysosomal integral membrane proteins—LIMPs
•lysosomal associated membrane proteins—LAMPs
I- Cell Disease (Inclusion- cell disease or mucolipidosis type II )
•Autosomal recessive disorder
•Caused by a mutation in the phosphotransferase responsible for converting mannose into M6P
•Proteins cannot associate with M6P-R and are not incorporated into vesicles derived from the TGN.
•Lysosomal contents are released into the extracellular fluid.
•Recall that proteins made on bound ribosomes are sent to the Golgi and are ultimately destined for the plasma membrane unless some other targeting sequence is found.
•The enzymes are not active at plasma pH 7.4.
•Provides a simple diagnostic test: If a sample of plasma contains a concentration of acid hydrolases, it indicates I-cell disease.
Clinical presentation of I Cell Disease
•Clinically, the patient with I-cell disease presents with failure to thrive, skeletal deformities, and abnormal facial features.
•Lysosomes are critical to the normal remodeling of cartilage and bone
•Hepatomegaly is also present, as well as clouding of the cornea (later in the disease) and chronic infection.
•There is no cure for I-cell disease.
•Children rarely survive the first decade of life.
•Many other lysosomal storage diseases also exist
•Characterized by a mutation in a single lysosomal enzyme, leading to the accumulation of a single type of molecule within lysosomes.
•All are autosomal recessive disorders and are more prevalent in the Ashkenazi Jewish population.
•Gaucher disease is the most common lysosomal storage disease.
•Due to a mutation in glucocerebrosidase
•Enzyme responsible for the hydrolysis of glucocerebroside (a sphingolipid)
•Lysosomes accumulate glucocerebroside
Clinically presentation of Gaucher's Disease
•hepatosplenomegaly, •osteoporosis, mental retardation, and seizures are complications
•Patients may not survive 5 years of age, or they may have a normal life expectancy, depending on the specific mutation and availability of treatment.
•Treatment is available for some forms.
•enzyme replacement (giving injections of glucocerebrosidase)
•inhibition of glucosylceramide synthase (the enzyme that synthesizes glucocerebroside)
•Tay-Sachs disease is probably the most recognized lysosomal storage disease.
•β-hexosaminidase A is defective
•responsible for the breakdown of GM2-ganglioside, another sphingolipid
•Symptoms present within the first 6 months of life and include progressive loss of hearing and vision, abnormal muscle tone, and paralysis.
•A "cherry-red spot" is seen on the retina.
•Most patients do not survive past 4-5 years old.
Clinically presentation of Tay- Sachs Disease
•Symptoms present within the first 6 months of life and include progressive loss of hearing and vision, abnormal muscle tone, and paralysis.
Neimann- Pick Disease
•Neimann-Pick disease -Results from a defect in sphingomyelinase, which is responsible for the hydrolysis of sphingomyelin, another sphingolipid.
Clinically presentation of Neimann- Pick Disease
•Clinical presentation includes hepatosplenomegaly, bone deformities, and CNS complications such as dystonia, seizures, and mental retardation.
•A "cherry-red spot" is also seen on the retina.
•These patients often do not survive their second birthday.
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