Organelles Flashcards
1
Q
Protein Import
A
all protein synthesis begins in the cytosol, but they are transported via three mechanisms:
- Transport via nuclear pores
- transport via protein translocators
- transport via vesicles
2
Q
Nuclear pores
A
located in the nuclear envelope function as selective gates that actively transport proteins in both directions between cytosol and nucleus
3
Q
Protein translocators
A
within organelle membrane directly transport proteins from the cytosol into the organelle. occurs co-translationally for ER and post-translationally for mitochondria and peroxiomes. the protein usually unfolds during transport
4
Q
Vesicles
A
moving proteins from one organelle to another via buds of membrane. vesicles containing proteins from the lumen and membrane of one organelle pinch off and fuse with the membrane of a second organelle. ER to Golgi, Golgi to PM, ECM, golgi to lysosomes. no vesicles to mitochondria or peroxiomes
5
Q
Signal Sequence
A
sorting signal that direct proteins to the right organelles. also called sorting signals or signal peptides. continuous stretch of AA (3-60) within protein, may be removed by peptidase once in organelle. functionally interchangeable (can take off one and put it on another). recognized by specific receptors.
6
Q
Mitochondria structure
A
matrix, inner membrane, outer membrane, intermembrane space.
7
Q
Matrix
A
large internal space. contains:
- citric acid cycle enzymes
- mitochondrial DNA
- replication/transcription/translation machinery
8
Q
Inner Membrane
A
cristae increase SA, e- transport chain, ATP synthase, transport proteins, electrochemical gradient
9
Q
Outer Membrane
A
porin forms channels for small ions and metabolites
10
Q
Intermembrane Space
A
Cytochrome c
11
Q
ATP Production in Mitochondria
A
pyruvate and FA from glycogen and fats go into make acetyl CoA, which goes into the citric acid cycle. That produces NADH high energy electron carrier. this passes electrons through the electron transport chain which makes an electrochemical proton gradient. electrons are transferred to oxygen to make water. when the proton goes back down gradient, ATP synthase makes an ATP from ADP and Pi.
12
Q
Mitochondria and Apoptosis
A
cytochrome c released from intermembrane space into cytosol, activates caspase cascade which leads to apoptosis.
13
Q
Protein transport into mito
A
signal sequence of precursor protein binds to TOM. TOM diffuses laterally to contact point (inner and outer membrane meet) and interacts with TIM. Chaperones help pull the protein through and fold it once it is inside. mitochondrial peptidase cleaves the signal sequence.
Transport of proteins to other sites within the mito require additional signal sequences that are revealed after the initial signal sequence is removed and additional membrane transporters.
14
Q
Mitochondrial Genome
A
very small circular double stranded DNA light strand and heavy strand
encodes 2 rRNAs, 22 tRNAs, 13 mRNAs
little regulatory sequence
no introns
genetic code is slightly different (codons mean different things than nuclear DNA)
~10-20 copies/ mitochondrion
15
Q
Mitochondrial Proteins
A
some come from mtDNA- 13 from the mRNA. the rest come from nuclear DNA and are transported to the mitochondria.
16
Q
Replication of mtDNA
A
origin of replication on each strand
replication occurs throughout the cell cycle
mtDNA chosen at random for rep
on average, number of mtDNAs doubles in each cell cycle.
17
Q
Transcription of mtDNA
A
both strands transcribed from single promotor on each
produces two giant RNAs, each a transcript of one DNA strand
RNA is cleaved into rRNA, tRNA, and mRNA
18
Q
Translation of mitochondrial mRNA
A
occurs in matrix
uses tRNAs and rRNAs encoded in mtDNA
produces 13 polypeptides- all subunits of complexes used in oxidative phosphorylation
19
Q
Peroxiomes
A
small multi functional organelles found in all eukaryotic cells. major site of oxygen utilization. the contain high concentrations of oxidative enzymes used in reactions that break down lipids and destroy toxic molecules. their size, number, and enzymatic content vary depending upon the cell type and metabolic needs of the organism. typical human cell as several hundred
20
Q
Oxidative degradation
A
remove hydrogen atoms from organic substrates and produces h2o2
RH2 + O2 –> R +H2O2
21
Q
Catalase
A
uses hydrogen peroxide to oxidize a variety of substrates such as phenols, formic acid, formaldehyde, and alcohol:
H2O2 +R’H2 –> R’ + 2H2O
kidney and liver cells use for detox
22
Q
Beta oxidation
A
long fatty acid chains to acetyl CoA
23
Q
Other F(x) of peroxiomes
A
synthesis of cholesterol, bile acids and some lipids
24
Q
Protein Import into Peroxiomes
A
post translational via translocators
25
Peroxiome disorders
Defective import: loss of f(x) empty peroxiomes or none. Zellweger syndrome. enzymes synthesized but not imported
Single protein defects: less severe. defect in only one thing..partially functional. X-linked adrenoleukodystrophy (ALD). lack membrane protein involved in long FA chain degradation. Leads to demyelination. Can do Allogenic stem cell transplant or gene therapy with HSC and HIV derived lentiviral vector expressing wildtype. eradicate bone marrow and add their own HSCs back in.
26
Endoplasmic Reticulum
single organelle consisting of interconnected flattened sacs and branching tubules of membrane. it is continuous with the outer nuclear membrane and typically extends throughout the cell. the ER lumen is the internal space enclosed by the ER membrane. subdivided into rough and smooth ER, rough has ribosomes
27
Functions of the ER
```
protein synthesis
protein modification
protein quality control
lipid synthesis
synthesis of steroid hormones
detox of lipid soluble drugs
ca2+ storage
```
28
Rough ER
entry point for proteins destined for other organelles or the plasma membrane. synthesizes proteins bound for the lumens or membranes of ER, Golgi, lysosomes or endosomes, plasma membrane, excretion to the cell exterior. once in the ER, proteins only travel out in vesicles and don't exit as single proteins (proteins synthesized in the cytosol stay there or may go into mito or peroxiomes with signals).
29
Protein Import into ER
occurs co-translationally. protein synthesis begins in cytosol, ribosome binds to mRNA (mRNA can bind to more than one ribosome at a time forming a polyribosome-then whole complex can bind to ER). ER targeting signal sequence is recognized by signal recognition particle (SRP) which stops translation momentarily while protein is transferred to ER. SRP binds to SRP-receptor and binds the ribosome to the translocation channel. SRP releases signal sequence and protein synthesis continues through the channel. SRP back to cytosol and signal peptidase can cleave the signal sequence.
30
SRP
signal recognition particle
31
Import of Soluble Proteins into ER
ER signal sequences almost always at N-Terminus. Signal sequence keeps translocation channel open during translation. protein is threaded through as a large loop. signal sequence cleaved by peptidase, which releases the protein into the lumen, chaperones help fold
Ex:
BiP, protein disulfide isomerase, other proteins that do modification in the ER
32
Import of Membrane Proteins into ER
have membrane spanning domains. ER targeting sequence may be at N-terminus or internally. when imported, some parts of the polypeptide chain are transported across the membrane while the membrane spanning domains are released laterally from translocation channel to become embedded in the ER membrane. This is determined by the stop and starts encoded in the mRNA that drives the protein synthesis (AA sequence directs lateral release), uses hydrophobic start transfer sequences. always inserted in one orientation so that the right domains are on the right side.
33
Protein Modifications in the ER
signal sequence cleavage, n-linked glycosylation, hyroxylation of collagen, protein folding and disulfide bond formation, assembly of multisubunit proteins
34
Signal Sequence Cleavage
signal sequences are cleaved co-translationally if they are n terminus by signal peptidases in the ER lumen
35
N-linked Glycosylation
proteins are converted to glycoproteins by the covalent addition of sugars. n-linked found on 90% of all glycoproteins. steps:
1. pre-formed oligosaccharide of 14 sugars is covalently attaches to Asp residues during translocation (co-translationally)
2. oligosaccharyl transferase (ER lumen), transfer the oligosaccharide block from dilochol (lipid in the ER membrane) to the polypeptide.
3. attached to the NH2 group
4. later modified by sugar removals and additions in ER and Golgi
36
Hydroxylation of Collagen
collagen molecules are hydroxylated on prolines and lysines to allow interchain H bonds to stabilize triple stranded helix of collagen molecules.
37
Protein Folding and Disulfide bond formation
chaperone proteins in ER help with folding. protein disulfide isomerase forms s-s bonds between cysteine side chains. stabilizes protein and helps it fold correctly (can't form in cytosol due to reducing environment)
38
Assembly of multi-subunit proteins
assembled with partner polypeptides in the ER. acetylcholine receptor is 5 separate polypeptides that must be assembled in the ER prior to transport to the cell surface.
39
Retention of ER Resident Proteins
KDEL at the C termini that retains them to the ER. They actually go to Golgi first then come back in vesicles. ER resident proteins. protein disulfide isomerase, oligosaccharyl transferase, signal peptidase.
40
Protein Quality Control in ER
Exit from ER is highly selective- if not folded right they can't exit. they bind to chaperone proteins and are retained in the ER until fixed, may eventually be degraded by proteasomes
41
Membran lipid synthesis in ER
produces nearly all lipids required for making new cell membranes, the secretory pathway, phosopholipids and cholesterol. most for mito and peroxiomes
the cytosolic half of the ER makes the new lipids via enzymes. added to the cytosolic part of ER. Flippases transfer them to other side so the membrane grows at a constant rate.
42
Membrane Lipid transport from the ER
via vesicles to Golgi, lysosomes, PM, endosomes
| via carrier proteins to mitochondira and peroxiomes
43
Smooth ER
synthesizes steroid hormones, detoxifies lipid soluble drugs, ca2+ sequestration. smooth ER can increase SA if increase in toxins such as alcohol
44
Calcium and the ER
release of calcium mediates intracellular signaling. SR releases calcium in response to AP to induce contraction
45
Vesicular Transport
transport proteins and lipids from the ER to the PM and from PM to lysosomes. Guided by proteins associated with the transport vesicle membrane. proteins have a target organelle
46
Secretory pathway
proteins synthesized in the ER are delivered to the cell surface or lysosomes via the Golgi
47
endocytic pathway
responsible for the ingestion and degradation of extracellular molecules; proteins are taken up at the PM and delivered to lysosomes via endosomes
48
Protein coat
most transport vehicles have protein coat on their cytosolic surface. vesicles bud from protein coated regions of membrane to form coated vesicles. coats are discarded prior to fusion with target. coat shapes membrane into bud and helps capture molecules for transport. clathrin is an ex.-golgi to secretory path and PM to endosomes as well
49
clathrin coated vesicles
assembles on cytosolic surface of trans Golgi membrane and forms a baseline membrane of hexagons and pentagons via 3 heavy and 3 light chains. looks like a soccer ball. induces curvature into the membrane in a pit which makes it easier to make a bud.
50
Adaptins
second major coat protein in clathrin coated vesicles. bind clathrin to the vesicle membrane and help select cargo molecules via its interaction with clathrin and transmembrane cargo receptors. adaptins vary depending on the nature of the cargo receptors
Clathrin-Adaptin-Receptor-Cargo
51
Dynamin
small monomeric GTP binding protein, assembles as a ring around the neck of each bud. Hydrolisis of GTP to GDP + Pi causes the ring to constrict, pinching it as a vesicle. clathrin coat removes after vesicle is pinched off
52
Actual Transport
after budding, vesicles are transported by motor proteins that move along microtubules. Transport vesicles have surface markers that display origin and cargo to be recognized by target membrane
53
Rabs
subfamily of momomeric GTPases that id each membrane type (on vesicle), they are recognized by tethering proteins on target membranes
54
tethering proteins
capture vesicles via their interaction with Rabs
55
SNAREs
v-SNAREs and t-SNAREs interact to dock vesicle. they also catalyze the fusion of the vesicle with the target membrane. fusion is a much closer association of membranes than docking, water must be displaced so need enzymes. SNAREs wrap around each other and pull the membranes close enough. Recycled after use
56
Exocytosis
new proteins and lipid are delivered from the ER, via the Golgi, to the cell surface and cell exterior by transport vesicles that fuse with the PM
57
Golgi apparatus structure
- flattened membrane bounded cisternae stacked together (3-20/Golgi stack).
- 2 distinct faces in each golgi stack
- cis (entry) and trans (exit) faces
- network of interconnected tubular and cisternal structures form the cis and trans Golgi networks at either end of stack
- usually located near the cell nucleus
- number of Golgi stacks per cell varies from one to many depending on cell type
58
Functions of Golgi
- sorting and dispatching station for proteins and lipids made in ER
- modification of N-linked oligosaccharide chains on glycoproteins made in ER
- synthesis of O-linked oligosaccharides on proteins and lipids made in ER
- synthesis of glycosaminoglycan chains on core proteins of proteoglycans
59
Protein Sorting
ER retention signals are sent back to the ER
| sorting occurs on both sides of the Golgi
60
Cis sorting
sort proteins that need to be returned to ER
61
Trans sorting
sorts proteins destined for lysosomes or regulated secretion from those that will continue to PM in DEFAULT PATHWAY
62
Default Pathway
ER-Golgi (modified if needed)-PM. these proteins initially only had ER target signal to get into ER to be synthesized.
63
Non-default
either go to lysosomes or are kept for regulated release
64
Processing Compartments
processing of adding sugars occurs in an organized sequence in the golgi stack with each cisterna containing its own enzymes. occurs in a spatial and biochemical sequence..early events have those enzymes in the cis part and changes as molecules move
65
Modification of N-linked oligosaccharides
carried out by glycosidases and glycosyl transferases..each step dependent on previous. can be changed to three major classes: high mannose, complex, hybrid
66
high mannose
most of the sugar stays the same..only take a little off
67
complex
changes almost whole backbone of sugar
68
hybrid
somewhere in the middle of modifications
69
O-linked glycosylation
covalent attachment of sugars to the OH groups of serine residues or threonine residues. sugars are added one and a time and build the sugar onto the protein. sugars are added post-translationally by glycosyltransferases
70
N-linked vs O linked
n=co translational, whole sugar at once, oligosaccharide transferase, from dilochol
o= post translational, adds sugars one by one, glycosyltransferase
71
Proteoglycans
contain at least 95% carbohydrate with a small protein core. carb always has glycosaminoglycan. variety of sizes with varying GAGs. Most GAGs attached on serine residue.
72
Glycosaminoglycans
linear polymers of a repeating disaccharide unit 100s of sugars long. high density of negative charges due to carboxyl and sulfate groups-can attract water and form gels. there are 6 classes, which differ in functions and locations
73
Function of Proteoglycans
lubricants (mucous secretions) and gels that can spring back when compressed (synovial joints and vitreous humor of eye).
74
Proteoglycan synthesis
- core protein synthesized in ER then transported to Golgi
- glycosyltransferases act sequntially to build a 4 sugar linker on serine of core protein
- repeated action of two specific glycosyltransferases adds repeating sugars of GAG chain
- sugars modified (Sulfation) as the chain grows
- core protein can also contain n-linked or o-linked oligosaccharides.
75
Constitutive Exocytosis Pathway
- default because all proteins will enter this unless otherwise specified.
- vesicles bud from the trans Golgi network and fuse with the PM
- operates continually in all cells and supplies PM with lipids and proteins
- responsible for constitutive secretions of proteins to the cell exterior
76
Regulated Exocytosis Pathway
in secretory cells
- proteins diverted into secretory vesicles which bud off from Golgi and accumulate near PM-aggregate due to special signals on membranes
- vesicles fuse with PM to release contents only in response to an extracellular signal
77
Endocytosis
cells take up fluid, macromolecules, particulates, and route them to lysosomes via endosomes.
78
Pinocytosis
ingestion of fluid and small molecules via small vesicles, occurs continually in all eukaryotic cells
79
Phagocytosis
- uptake of large particles such as bacteria in large vesicles
- requires receptor activation at cell surface
- occurs in specialized cells (macrophages and neutrophils)
80
Phagocytosis
macrophage and neutrophils. once induced by receptors, cell extends projections of its PM to engulf microorganism, forms a phagosome. Phagosomes fuse with lysosomes and material is degraded. Macrophages eat a lot of RBCs, ant apoptotic cells
81
Pinocytosis 2
carried out by clathrin coated pits and vesicles (pit forms from clathrin binding and interaction with adaptins and causes bud to form). pits invaginate cells and pinch off to form vesicles. clathrin sheds and vesicle fuses with early endosome. Extracellular fluid is delivered as well.
82
Receptor Mediated Endocytosis
macromolecule binds to a specific cell surface receptor in the PM which then accumulates in coated pits and is internalized in clathrin coated vesicles--then taken to endosome to be sorted or taken somewhere else
83
Example of Receptor Mediated-Uptake of Cholesterol
- cholesterol is transported by LDL
- cells make LDL receptors and put them in PM
- LDL receptors associate with clathrin pits via adaptins and are internalized into vesicles along with and LDL bound to the receptors.
- Vesicles shed coats and fuse with early endosomes- acidic environment causes LDL to break from receptor
- LDL transported to lysosomes and hydrolyzed to free cholesterol which is released into cytosol
- LDL receptor recycled if needed
84
Familial Hypercholesteremia
defect in LDL receptor gene-leads to super high levels of cholesterol in blood.
85
Early endosomes
located near PM
- act as sorting station for endocytic pathway
- endocytosed materials appear here within a minute of uptake
- acidic environment (pH ~6) allows some receptors to release their ligands
86
Late endosome
located near nucleus
- endocytosed materials arrive 5-15 min after uptake
- materials ultimately transported to lysosomes via vesicles or gradual conversion of endosome to lysosome.
87
Pathways from endosomes
1. Recycling- return to PM (LDL receptors)
2. Degredation- early endosome to lysosome--dec in receptor concentration= receptor down-regulation
3. Transcytosis- return to a different PM domain in polarized cells (receptor antibody complexes in breast epithelium) bloodstream to milk-endocytosed at basolateral and delivered to apical membrane
88
Lysosomes
- intracellular digestion
- contains hydrolases to digest extracellular materials and worn out organelles
- membranes have ATP driven pump that maintains the lumen at an acidic pH of 5
- membranes contain transport proteins that transport digestion products to cytosol
- membrane proteins heavily glycosylated to protect them from proteases
89
Delivery of Lysosome enzymes to lysosome
mannose-6-phosphate (M6P) tag is added to lysosomal enzymes in Golgi which directs them to special transport vesicles in trans Golgi for delivery to lysosomes via endosomes
90
Material Delivery to Lysosome
1. endocytosis
2. Phagocytosis- phagosomes fuse with lysosomes
3. autophagy- digestion of obsolete cell parts. a double cell membrane surrounds an autophagosome which then fuses with late endosome/lysosome
91
Lysosomal storage disease
genetic defects affect hydrolases-accumulation of undigested substances
92
Mucopolysaccharidoses
genetic diseases caused by defects in the enzyme required for degradation of GAGs. Partially digested GAGs accumulate in lysosomes and blood and CT. permanent progressive cellular damage. after cell death materials appear in urine.
93
Oligosaccharidoses
genetic disease in hydrolases for degredation of oligosaccharides. phenotypes vary depending on enzyme affected. as above with partially digested and urine
94
Inclusion cell disease (I-cell)
rare lysosomal storage disease in which almost all of the enzymes are missing, defect is in the enzyme responsible for generating M6P markers on hydrolases. cant get to lysosome, secreted via default pathway. undigested substrates accumulate in lysosomes and form large inclusions.
95
Glycoprotein
Any protein with one or more covalently bound carbohydrate units that do NOT contain a serial repeat
(i.e. the carbohydrate is not a glycosaminoglycan). Typically, but not always, they are mostly protein
with a little carbohydrate attached.
• mass of carbohydrate varies from 2 to 80%
• number of covalently linked carbohydrates varies from 1 to 100s
• number of sugar residues in each carbohydrate varies from 1 to 15
• carbohydrate may be covalently bound to the amino group of Asn (N-linked) or the hydroxyl
group of Ser or Thr (O-linked)
• structure of the carbohydrates on a single protein can be very different from each other creating
extensive microheterogeneity
synthesized in ER--membrane proteins have carbs on lumenal sides, which is the same side as ECM in proteins in PM
96
Proteoglycan
Any protein with one or more covalently bound glycosaminoglycan chains. Typically, but not always,
they are mostly carbohydrate with a very small core protein.
• mass of carbohydrate usually ≥ 95%
• core protein is very small
97
Gycosaminoglycan
Linear polymer of a repeating disaccharide unit (uronic acid (or galactose) – hexosamine)n. These chains
are typically 100s of sugars long and carry many negative charges on the carboxyl groups of the uronic
acids and on sulfate groups which are added to many of the sugars. (Example: heparin)
98
Oligosaccharide
3-15 sugars covalently bound in a linear or branched chain (ex: ABO blood group antigens)
99
Glycosidic bond
Covalent bond between two monosaccharides
100
Polysaccharide
Long linear or branched polymer of sugars (ex: glycosaminoglycans, glycogen, starch)
101
Glycosidases
Enzymes that cleave glycosidic bonds
102
Glycosyltransferases
Enzymes that form glycosidic bonds; they transfer a monosaccharide from a nucleotide sugar (i.e. UDPglucose) to a growing oligosaccharide or polysaccharide.
103
Oligosaccaryltransferase
Enzyme that transfers a pre-formed oligosaccharide from its lipid anchor (dolichol) to an appropriate Asn
residue in a polypeptide during N-linked glycosylation in the ER.
104
Functions of Glycoproteins
* Structure and support in the extracellular matrix (ex: collagen, fibrin)
* Hormones (ex: follicle stimulating hormone, chorionic gonadotropin)
* Lubrication and protection (ex: mucins, mucus secretions)
* Enzymes (ex: lysosomal hydrolases, proteases)
* Immunologic molecules (ex: immunoglobulins, complement)
* Cell surface antigens (ex: ABO blood group antigens)
* Plasma proteins (ex: blood clotting proteins)
105
Mucins
Macromolecule in mucous, which are synthesized and secreted in the epithelium.
viscous glycoprotein composed of 80% carb by mass. atypical--much more carb than protein. carb consists of O-linked oligosaccharides of varying structure, and some N-linked.
-rod shaped proteins with a central region enriched with Ser and Thr to allow for high O-Linked sugars.
-other regions are enriched in Cys, which allows polymerization of molecules via s-s bonds. generate hydrated gel resistant to proteases
106
ABO blood group antigens
- oligosaccharide
- antigens are oligosaccharide components of O-linked glycoproteins and glycolipids found on the surface of red blood cells.
- determined by single gene locus (ABO), which encodes a glycosyltransferase responsible for transferring a terminal sugar to galactose on the O antigen (every antigen starts as O- is modified to become A or B by glycosyltransferase)
- product of ABO locus determines if a sugar will be added to O antigen
107
A allele
encodes a transferase that transfers an N-acetyl galactosamine (GalNAc); codominant with B allele
108
B allele
encodes a transferase that transfers a galactose (Gal) differs from A by 4 aa; codominant with B allele
109
O allele
non-functional protein due to single nucleotide deletion and frame shift
110
Codominance
neither allele is dominant over the other. if both are present, both transferases will be present and RBCs will have A and B antigens
111
Type O
OO- make neither transferase. only O antigen. no A or B to be recognized by other immune systems, but have anti a and anti b antibodies
112
Type A
AA or AO- GalNAc on RBCs so only A antigen. have anti B antibody
113
Type B
BB or BO- only Gal and B antigens. anti a antibody
114
Type AB
AB-RBCs have A and B antigens- no antibodies
115
Degradation of Glycoproteins and Proteoglycans
lysosomes via lysosomal hydrolases (proteases and glycosidases)
endoglycosidases remove carbs
proteases cleave protein into aa
glycosidases act on each glycosidic bond in reverse order that they were added
additional enzymes remove other chemical groups-sulfates and acetyl groups
