Cell Biology - The Endomembrane System

Front 1

What does the Endomembrane System consist of?

Back 1

Nuclear envelope and perinuclear space

endoplasmic reticulum

golgi complex

endosomes (anything phagocytosed into a cell)

lysosomes

transport vesicles

Front 2

What is the Peroxisome and the ER?

Back 2

Peroxisome:
-single membrane bound organelle
-generation and degradation of H2O2
-contains catalase (destroys H2O2)
-detoxification
-breakdown of long chain FA
-lots in the Liver

ER:
-has sacs calls cisternae
-space between sacs is called lumen
-site of biosynthesis of lipids and secreted proteins

Smooth:
-lack ribosomes
-drug detoxification in liver by making them less hydrophobic
-hydroxylation (steroid biosynthesis)
-transfers electrons from NADH to cytochrome P-450

Rough:
-synthesize secretory proteins, lysosomes, and plasma membranes
-transitional elements = shuttle lipids and proteins from ER to golgi
-site for addition of carbs to glycoproteins, folding of polypeptides, and assembly of multimeric proteins
-Protein quality control = ER associated degradation (ERAD), abnormal proteins exported by cytosolic proteosomes

Front 3

What is cytochrome P-450 and P-448?

Back 3

P-450:
-activates oxygen for hydroxylation reactions in the smooth ER
-enzymes that are activated are called mixed-function oxidases or monooxygenases
-example of monooxygenase is elimination of barbiturate drugs

P-448:
-hydrocarbon hydroxylases
-another group of enzymes that catalyze the hydroxylation reaction
-metabolize polycyclic hydrocarbons
-cigarette smoke is a potent inducer of aryl hydrocarbone hydroxylase

Front 4

What are some specializes functions of the Smooth ER?

Back 4

Carbohydrate metabolism:
-in liver cells contains glucose-6-phosphatase which dephosphorylates G-6-P to glucose
-without this phosphatase glucose cannot be formed from glycogen

Calcium Storage:
-Calcium ions pumped into ER by ATP-dependant Ca pumps in SR
-ex. SR found in muscle cells

Front 5

What does the Golgi Complex play an important role in and what are its components?

Back 5

1. glycosylation of proteins, sorting of proteins for transport to organelles, plasma membrane, or for secretion

2. transition vesicles from ER fuse with cis-Golgi network (CGN) delivering lipids and proteins to golgi complex, eventually goes to Trans-Golgi Network (TGN)

Components:
-two faces of Golgi stack: cis (facing ER) and trans (tip of TGN cisternae)
-Medial cisternae: between CGN and TGN, NAGT1 is concentrated here
-stacks are biochemically DISTINCT

Front 6

What are transport and coated vesicles?

Back 6

Transport vesicles:
-transport of lipids and proteins

coated vesicles:
-ER to CGN coated with COP II
-CGN to medial coated with COP I
-TGN coated with either COP I or clathrin transport vesicles

Front 7

What are the two models that depict the flow of lipids and proteins through the Golgi Complex?

Back 7

Stationary Cisternae Model:
-each part of the Golgi stack is a stable structure and traffic is mediated by shuttle vesicles that bud from one cisterna and fuse with another

Cisternal Maturation model:
-gradually change from CGN to medial to TGN
-supported via labelling

Front 8

What are anterograde and retrograde transport?

Back 8

anterograde:
-movement from ER through Golgi toward Plasma membrane

retrograde:
-vesicles from Golgi cisternae to ER

Front 9

What are the stages of the Role of ER and Golgi in Protein Synthesis?

Back 9

1st stage:
-start attached to ER in cytosol, then get flipped into lumen = CORE GLYCOSYLATION
-Nlinked = Asparagine, Olinked = Serine or Threonine
-core structure of carbs consist of two GlcNAc units, 9 mannose, 3 glucose
-Protein is NOT HERE YET

2nd stage:
-in ER: core oligosaccharide is trimmed, 3 glucose and 1 mannose by glucosidase and mannosidase
-in Golgi: further modifications
-if no further processing = high mannose oligosaccharide
-processing = complex oligosaccharides (ex. galactose, sialic acid, fucose, N-acetyl)
-over 200 glycosyl transferases in ER and Golgi

Following biosynthesis, glycosylated proteins and liipds face interiors of ER and Golgi

Front 10

What are the steps of Glycosylation and modification of proteins from ER through Golgi?

Back 10

In ER:
-biosynthesis of core oligosaccharide for N-linked asparagine residues
-processing of core oligosaccharides
-ID and removal of misfolded proteins

CGN:
-attachment of N-acetylgalactosamine to serine or threonine
-first step of phosphorylation of lysosomal proteins

CGN --> Medial Cisternae
-removal of mannose
-2nd step of phosphorylation of lysosomal proteins

Medial Cisternae:
-removal of mannose
-attachment to N-acetylglucosamine

Medial Cisternae --> TGN
-addition of galactose
-addition of sialic acid

TGN:
-addition of sialic acid
-attachment of sulfate to tyrosine

Front 11

How does Glycosylation help promote proper protein folding?

Back 11

inhibition leads to misfolded proteins

during 2nd stage when glucoses are removed, ER proteins calnexin (membrane bound) or calreticulin (soluble) complex with a glycoprotein and a thiol oxidoreductase (ERp57) to promote disulfide bond formation

UGGT then acts as sensor for proper protein folding (quality control), if improper will complex back with calnexin/calreticulin

Front 12

How do membrane lipids mark vesicles?

Back 12

tag is phosphate group added to posphatidylinositol (PI)

PI-3 kinase is required

inhibition of kinases perturbs vesicle trafficking to the lysosomes

Front 13

What is the trafficking process through the endomembrane system?

Back 13

1. Proteins synthesized by ribosomes, initial glycosylation steps occur within ER lumen

2. transition vesicles take new lipids and glycosylated proteins to CGN

3. Lipids and proteins move through golgi stack. At TGN vesicles form either secretory vesicles or endosomes

4. Materials taken in by endocytosis, fuse with early endosomes

5. Unused materials recycled to plasma membrane

6. endosomes become lysosomes

7. retrograde traffic takes stuff back

Front 14

What are Retrieval tags for ER proteins?

Back 14

KDEL:
-when protein bound to retrieval tag it goes through conformational change and returns to ER
-because acidity lower in ER than CGN protein dissociates
-matures in Golgi
-KDEL is a ligand for the receptor so becomes bound to be able to come back to ER (retrieval and receptor mediated transport)

RESONANT proteins stay in ER, too big for transport and are hydrophobic, therefore dont need to go to Golgi for maturation

Front 15

What are properties of length for golgi complex proteins?

Back 15

increase in length of hydrophobic spanning domains from CGN to TGN

membrane thickness increases from ER to plasma membrane

Front 16

How are proteins targeted for lysosomes sorted?

Back 16

in ER, N-glycosylation leads to removal of glucose and mannose

in golgi: N-acetylglucosamine-I-phosphate is added to C6 of mannose by the first enzyme, second enzyme removes N-acetyle glucoasmine, leaving the mannose residue

Lysosomal enzyme is synthesized and carbohydrate is added

mannose is phosphorylated by sequential activity of two enzymes

mannose-6-phosphate binds to receptor and tagged enzymes are packaged in clathrin coated transport vesicles

pH is lowered, enzyme dissociated from receptor

receptors are recycled in vesicles that return to TGN

Front 17

What is I Cell disease?

Back 17

mucolipidosis

fibroblasts release lysosomal enzymes extracellularly

-defective phosphotransferase leads to lack of mannose-6-phosphate tags
-however other pathways exist for targeting lysosomes
-in disease RECEPTOR FOR MANNOSE TAG IS EXCRETED

Front 18

What are two Secretory pathways to transport molecules to exterior of cell?

Back 18

Constitutive secretion:
-vesicles bud from TGN and move directly to cell surface, process is continuous and independant
ex. mucus cells (all the time)

Regulated secretion:
-vesicles accumulate int he cell then fuse with plasma membrane only in response to extracellular signals
ex. Pancreatic insulin cells

Front 19

What happened when a radiolabeled AA added to Rabbit?

Back 19

tracer kept building, so there was no secretion due to lack of tags, problem with secretory vesicles

Front 20

What happens in Endo, Exo, and phagocytosis?

Back 20

Exocytosis:
-calcium triggers exocytosis (activation of kinases)

Endocytosis (pinocytosis):
-2 ways: non-specific clathrin independant, receptor mediated

phagocytosis:
-large particles
-human phagocytes (neutrophils)
-fibroblasts take up collagen to help CT
-generate H2O2 to kill microorganisms
-takes some microorganism and sends it back to present on MHC for B cells to make antibodies
-pseudopod --> phagocytic vacuole --> fuses with early endosome --> forms into a mature lysosome --> digestion occurs

Front 21

What is receptor-mediated endocytosis and transcytosis?

Back 21

clathrin dependent endocytosis

-recycling of receptors by change in pH
-fuse with vesicles budding from TGN to form early endosomes --> acquire lysosomal proteins to mature into late endosome
ex. LDL receptors

transcytosis: material in vesicle moves to opposite side of cell and fuses with plasma membrane, releasing material into extracellular space

Front 22

What are Clathrin coated vesicles composed of?

Back 22

help with forming vesicles with flat membranes and targeting interactions with cytoskeleton

surrounded by lattices composed of clathrin and adaptor protein (AP), latlice composed of triskelion (heavy and light chains)

Adaptor protein - confer specificity and mediate attachment of clathrin

clathrin coat drives formation of vesicles from PM to TGN

dynamin: GTPase required for coated pit constriction and closing of budding vesicle

uncoating ATPase uncoats vesicles after they bud from membrane

Front 23

What is Clathrin independent Endocytosis?

Back 23

fluid phase endocytosis

indiscriminate uptake of material

primary mechanism of membrane recycling --> controls cell volume and surface area

Front 24

What are the different coats, where are they found, and what are some coat proteins?

Back 24

Clathrin: TGN --> Endosomes

Clathrin: Plasma membrane --> endosomes

COPI: Golgi complex --> ER or Golgi (retrograde)
-fuzzy coat
-do NOT bud from ER

COPII: ER --> Golgi

Caveolin: Plasma membrane --> ?

Proteins:
ARF: designated ADP ribosylation factor 1, switches GDP for GTP, inserts hydrophobic tail into membrane

AP1 and AP2 - designate different adaptor proteins

Front 25

What is the SNARE hypothesis and some examples?

Back 25

explaining how membrane vesicles fuse with the proper target membrane

specificity comes from vesicle-SNAREs, target-SNAREs, Rab GTPases and tethering proteins

SNAPs mediate fusion, NSF uncouples SNARE after fusion

best studied SNARES:
-botulism and tetanus

Front 26

What are tethering proteins?

Back 26

act over long distances

coiled-coil proteins:
-Golgins - needed for initial interaction of COP I and COP II coated vesicles to the Golgi
-also connect Golgi cisternae together

Multisubunit complexes:
-COG
-GARP
-TRAPP

Front 27

What are properties of Lysosomes?

Back 27

cellular digestion:
-degrade all classes of macromolecules
-acid hydrolases - keep low pH to help denature macromolecules

Endosomes:
-early endosomes = TGN + PM
-late endosomes have acid hydrolase
-final step is activation of hydrolases: by lowering pH, transferring material from late endosome to lysosome

Front 28

What are the two models of transfer from endosomes to lysosomes?

Back 28

Transient fusion model:
-late endosome forms temporary connection with lysosome
-only new proteins and lipids to be degraded are transferred

hybrid model:
-late endosome and lysosome fuse, with proteins and lipids not segregated

Front 29

WHat are different types of lysosomes?

Back 29

Heterophagic - extracellular origin

Autophagic - intracellular origin

phagocytosis and receptor-mediated endocytosis - lysosomes in defense and nutrition

residual body - indigestible remain in lysosome, contributes to cellular aging

autophagy:
macro - structure wrapped in double membrane from ER
micro - single membrane

extracellular digestion:
ex. sperm
-rheumatoid arthritis inadvertent release of lysosomal enzymes

Front 30

WHat are some lysosomal storage diseases? What is a peroxisomal disorders?

Back 30

Lysosomal:
Gaucher's disease:
-glucocerbrosidase defiiciency
-macrophages accumulate leading to liver and spleen enlargement

Type II glycogenosis (Pompes):
-excess glycogen in liver
-defective alpha 1,4 glucosidase

Hurler and Hunter:
-defect in degradation of glycosaminoglycans

Tay-Sachs:
-missing beta-N-acetylhexosimanse
-GM2

Sanfilippo A:
-Heparan sulfamidase

Niemann- Picks:
-Shingomyelinase

Krabbe's:
-beta galactosidase

Peroxisomal:
X-linked adrenoleukodystrophy:
-defective integral protein responsible for transporting very long chain FA
-destroys myelin sheath in CNS

Infantile Refsum disease:
-phytanic acid leading to build up of unsaturated FA

Zellweger's syndrome:
-congenital disorder, absence of peroxisomes in liver, kidneys, and brain

Neonatal adrenoleukodystraphy:
-X-linked (affects boys)
-FA accumulate in CNS and destroy myelin sheaths

Front 31

What are some functions of Peroxisomes?

Back 31

1. detoxification of harmful compounds

2. oxidation of long chain FA - beta oxidation

3. metabolism of nitrogen-containing compounds - prevents urate crystals which cause gout, catabolizes xenobiotics

4. catabolism of unusual substances

5. breakdown of hydrogen peroxide by catalse

6. urate oxidase breaks down purines (DNA)

7. enzymes for bile acid synthesis

Front 32

How is Hydrogen peroxide metabolized?

Back 32

catalytic mode:
-one molecule of hydrogen peroxide is oxidized, 2nd is reduced

peroxidatic mode:
-electrons derived from organic donor
ex. methanol, ethanol etc

Front 33

What are the single-membrane bound organelles?

Back 33

lysosome, peroxisome, and vacuoles

Front 34

Who are some nobel prize winners and what are they for?

Back 34

Gunter Blobel - proteins have intrinsic signals that govern their transport and localization in the cell (physiology)

Mendel - Genetics

PCR - Mullis

Prions - Prusiner

Hunt, Hartwell, Nurse - Cell Cyle

Front 35

What are proteosomes and ubiquitin?

Back 35

proteosomes:
-predominant protease in the cytosol

ubiquitin:
-targets proteins for degradation by proteosomes
-small protein chain

Front 36

What is the Ubiquitin-Proteosome pathway?

Back 36

Ubiquitin attaches to E1 (taxi)

Ubiquitin is then transferred to E2

E2-Ubiquitin plus E3 bind to a target protein

Ubiquitin molecules are attached to lysine residues in target proteins, E2 and E3 detach

Front 37

What are properties of ribosomes?

Back 37

40s (small), 60s (large) subunit

40s binds to mRNA and tRNA ands start codon AUG

60s binds to 40s after it finds start codon and has peptidyl transferase activity

protein syntheis - translation of mRNA into AA sequence

form polysome that is involved in synthesis of cytoplasmic proteins

cleave hydrophobic signal sequence by signal peptidase

Front 38

What are properties of CD8 and CD4 T cells and MHCs?

Back 38

CD8:
-proteasome pathway
-cytosol-derived antigen
-intracellular infection (virus)
-use granzymes and perforin to kill
-MHC I

CD4:
-lysosomal pathway
-Vesicle derived antigen
-extracelullar infection (bacteria)
-MHC II

MHC I - all nucleated cells
APC = MHC I and MHC II

MHC II deficiency - BARE LYMPHOCYTE SYNDROME

Front 39

What is AIRE?

Back 39

Master gene involved in self tolerance of the immune system

Autoimmune polyendocrinopathy:
-autoimmune destruction of endocrine glands caused by mutation in autoimmune regulator gene AIRE

AIRE is normally expressed in the thymus and serves as a transcription factor in thymic epithelial cells

Front 40

What is EAE?

Back 40

Experimental autoimmune encephalomyelitis:
-mouse model of organ specific autoimmune disease mediated by T lymphocytes
-model of MULTIPLE SCLEROSIS (destruction of myelin sheath)

Front 41

What are the Hypersensitivity Reactions?

Back 41

Type I: immediate hypersensitivity (mast cells and IgE)

Type II: reaction to drugs (penicillin)

Type III: Antigen-Antibody complex deposition

Type IV: delayed hypersensitivity

Front 42

What are the types of Immunodeficiencies?

Back 42

Primary:
-congenital

Secondary:
-example is HIV (targets CD4)

Front 43

What is hematopoesis?

Back 43

development and production of highly differentiated cells from precurso stem cells

cytokines help initiate

pluripotent stem cells give rise to all cells found in the blood

two lineages - myeloid and lymphoid

dendritic are from both lineages

Front 44

What are major APCs?

Back 44

dendritic cells (most professional)

macrophages

B cells