Term
| Describe the membrane bound organelles |
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Definition
-besides nucleus, eukaryotes have membrane bound organelles that allow them to function properly even tho they are 1000x larger than bacteria
-sorting and targeting proteins are important task
-accomplished by ER, Golgi, endosomes, lysosomes |
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Term
Describe the ER
1. Brief description
2. 3 domains |
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Definition
1. network of membrane enclosed tubules and sacs (cisternae) that extend from nuclear membrane
-largest organelle and membrane in continuous
2. Rough ER- covered by ribosomes
Transitional- vesicles exit to the Golgi
Smooth- no ribosomes and is involved in lipid metabolism |
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Term
Describe the secretory pathway
1. Discovery
2. Components |
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Definition
1. radio-labeled AA were incorporated into proteins and their location could be determined by autoradiography
-radioactive 35Sulfur was added to methionine
2. rough ER (protein synthesis) > Golgi> secretory vesicles > cell exterior
*not just secretory proteins (plasma membrane and lysosomal) |
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Term
| Describe Cotranslational translocation |
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Definition
-proteins are translocated to ER during synthesis on ribosomes
-saves energy!
-no need to cross membrane
-no localization signals
Posttranslational translocation: proteins are taken to ER after synthesis on free ribosomes
-require signals
ex. nucleus, mito, chloro, perox |
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Term
| Describe targeting proteins for ER: signal sequences |
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Definition
-all protein synthesis starts on free ribosomes
-polypeptide will contain signal sequence that will bring ribosome to ER
-sequences are located at amino terminus of polypeptide
-hydrophobic stretch of 20 AA
-usually cleaved after entrance into ER |
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Term
| Describe targeting proteins for ER: Cotranslation |
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Definition
Signal recognition particle
1. as sig seq emerges from ribosome, SRP binds in cytoplasm
-SRP contains 6 polypeptides and RNA
2. SRP binds to ribosome and sig seq, inhibiting translation
3. SRP binds to SRP receptor on ER
-releases SRP from ribosome and sig seq
4. sig seq is inserted into translocon membrane channel and translation resumes
5. sig seq is cleaved by signal peptidase and rest of polypeptide is released into lumen of ER
*coordinated by GTP hydrolysis |
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Term
| Describe targeting proteins for ER: Posttranslation |
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Definition
1. protein is fully synthesized in cytoplasm
2. chaperones (Hsp70) attach to protein to keep them unfolded so they can enter translocon
-signal sequence recognized by translocon complex
3. another Hsp70, BiP is present in ER and acts as ratchet to pulls polypeptide chain through channel |
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Term
Describe membrane proteins
1. Integration
2. Composition
3. Orientation |
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Definition
1. initially inserted directly into membrane
-transported along secretory pathway as membrane components
2. membrane spanning regions are normally alpha helix with hydrophobic AA
3. either N or C orientation
-multiple spanning regions have alpha helix followed by triple lysine that loop outside of membrane |
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Term
| Describe composition of ER lumen and protein orientation |
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Definition
ER and Golgi lumen = outside of cel (ECM)
-proteins on lumen side stay on lumen side until incorporated into plasma membrane where they orient on ECM side
-if on cytoplasmic side, they stay cytoplasmic and when they reach plasma membrane they incorporate to face cytoplasmic side |
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Term
Describe insertion of membrane proteins into ER membrane
Simple C terminus |
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Definition
1. similar to secreted proteins, sig seq is cleaved by signal peptidase and translation continues
2. a second alpha helix is synthesized called stop-transfer seq, halts translation
3. causes translocon to dismember and protein moves into membrane
4. translation continues with C terminus remaining in cytoplasm |
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Term
Describe insertion of membrane proteins into ER membrane
No cleavage |
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Definition
1. sig seq is translated but not cleaved
2. acts as hydrophobic membrane spanning region
-leaves translocon and translation resumes
3. depending which way seq was originated, either C or N terminus can be in cytoplasm |
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Term
Describe insertion of membrane proteins into ER membrane
Transmembrane |
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Definition
1. sig seq acts as hydrophobic spanning region and enters membrane
2. proteins translated until stop-transfer seq is encountered
-enters membrane
3. proteins translated until another sig seq is formed and enters back into translocon
4. process is repeated alternating sig seq and stop-transfer |
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Term
| Describe protein folding into ER |
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Definition
-can either by done during translocation or after
BiP (Hsp70)
1. binds to unfolded polypeptide as it crosses membrane
2. mediated folding and assembly by disulfide bonds |
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Term
| Describe the environment of the ER/Cytoplasm |
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Definition
ER= oxidizing
SH-> S-S
-formation of disulfide bonds to properly fold proteins
*Protein Disulfide isomerase (DPI) forms S-S
Cytoplasm = reducing
-keep in SH |
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Term
| Describe protein modifications in the ER |
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Definition
1. cleavages of signal sequences
2. protein folding (chaperones)*
3. assembly of multisubunit proteins (BiP)*
4. disulfide bond formation (PDI)
5. glycosylation
6. addition of glycolipid anchors to plasma membrane proteins |
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Term
| Describe protein glycosylation in ER and addition of glycolipids |
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Definition
Glycosylation
-proteins are glcosylated on Asparagine (N-linked) as they are translocated
-oligo is synthesized on dolichol phosphate carrier
-helps prevent protein aggregation and provides signal for subsequent sorting
Glycolipids
-GPI anchors attach proteins to membrane
-synthesized in ER and attached after completion to C terminus
-proteins remain outside of cell on plasma membrane |
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Term
| Describe protein quality control: Calreticulin |
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Definition
-assists in folding glycoproteins
1. calreticulin binds to proteins and assists folding
-1 glucose is removed and is released
2. checked by protein folding sensor
IF good: goes to transitional ER
IF real bad: marked for degradation by ubiquitin
IF incorrect: glucose readded by UDP-glucose and cycles back to calreticulin |
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Term
| Describe protein quality control: BiP |
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Definition
-acts as chaperone and sensor of protein folding
1. if excess of unfolded proteins, BiP binds to unfolded proteins and BiP no longer binds to signal molecules
-unfolded protein response
2. releases molecules that signal unfolded protein response
a. inhibition of protein synthesis
b. increased expression of chaperones
c. increased degradation of mRNAs |
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Term
| Describe lipid synthesis in smooth ER: Brief description |
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Definition
-most lipids are synthesized in ER
-lipids are hydrophobic so they are synthesized in association with already existing cellular membranes
-transported from ER to final destination
-new lipids synthesized on cytosolic side
-use CMP and CDP for energy
-smooth ER is abundant in cells active in lipid metabolism
ex. testis and ovaries
ex. liver |
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Term
| Describe lipid synthesis in smooth ER: Flippase |
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Definition
-phospholipids are synthesized on cytosolic side of ER from water soluble glycerol
-allows hydrophobic FA to remind buried in membrane
-new lipids are only made on outside so they must be transferred to other side to evenly produce new membrane
Flippase: facilitates passage of polar head through membrane
-cover polar head so it can pass through nonpolar
-flippases are specific to each kind of lipid |
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Term
| Describe how ER resident proteins remain in ER |
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Definition
ER resident proteins: BiP, PDI, signal peptidase
-targeting sequence of KDEL or KKXX at C terminus
-does not prevent but instead retrieves from Golgi
1. bind to specific receptors that are pH sensitive
Golgi= acidic ER= basic
2. bind in Golgi and then vesicle is taken back to ER
3. once in ER lumen, protein is released because its basic |
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Term
| Describe ER protein/lipid export |
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Definition
-bud from ER to transitional ER to ERGI then to Golgi
-proteins are marked with export sig seq
-either di-acidic or di-hydrophobic AA |
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Term
Describe the Golgi
1. Function
2. Structure |
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Definition
1. proteins from ER are processed and sorted for transport
-glycoplipids and sphingomyelin are synthesized
-complex cell walls in plant cells
2. flattened membrane enclosed sacs (cisternae)
a. cis- proteins enter from cis side toward ER and nucleus
b. medial and trans- modification done here
c. trans- exit from concave trans face |
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Term
| Describe glycosylation in the Golgi: N-linked |
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Definition
-N-linked oligos are modified in Golgi that were added in ER
-mannose are removed and N-acetylglucosamine, galactose, sialic acid are added
-wide variety of combinations
Glycotransferases add sugars
Glycosidases remove sugars (mannose) |
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Term
| Describe oligos for lysosome proteins |
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Definition
-N-linked oligos on lysosome proteins are modified by mannose phosphorylation (UDP phosphorylates)
1. enzyme recognizes structural determinant in 3D shape of protein
2. adds N-acetlyglucosamine phosphate
-removes NAG
3. leaves mannose-6-phosphate that binds to receptor in trans Golgi that sends it to endosome and then lysosome
*recognized by signal patches on lysosomal protein instead of AA sig seq |
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Term
| Describe glycosylation in the Golgi: O-linked |
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Definition
-added to Serine and Threonine
-usually linked to N-acetylgalactoseamine |
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Term
| Describe transport from Golgi to cell surface |
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Definition
1. Direct transport
-incorporation of new lipids into membrane
-continuous secretion
2. Recycling endosomes
3. Regulatory secretions
-controlled secretion in response to environmental signals
-proteins have patch signals that are recognized by cargo receptors
-packaged into special secretory vesicles
-stored and released when outside signal directs fusion with plasma membrane
ex. neurotransmitters, saliva, endocrine, insulin |
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Term
| Describe secretion in polarized cells |
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Definition
-plasma membrane is divided into apical domain and basolateral domain each with specific proteins
-proteins must be packaged accordingly and directed to correct membrane
ex. stomach epithelium- HCl, proteases |
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Term
| Describe the mechanism of membrane fusion |
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Definition
*mechanical process
-transport vesicles are coated with cytosolic coat proteins
-coats assemble as it buds and are removed before it fuses
-vesicles fuse with target and incorporate into membrane
-water prevents fusion
-vesicle must get close enough to dispel water so fusion can occur |
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Term
| Describe vesicle coat proteins |
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Definition
1. COP 1- bud from ERGIC or Golgi and go back to ER and return proteins to earlier compartments
2. COP 2- carry secretory proteins from ER to ERGIC
3. Clathrin- transport in both directions between trans Golgi and to other destinations
ex. Golgi, plasma membrane, endosomes, lysosomes |
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Term
| Describe clathrin coat assembly |
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Definition
3 requirements:
1. clathrin 2. GTP-binding protein ARF-1 3. adaptor proteins
-assembles into basket like structure that distorts membrane and initiates bud
-M6P binds to M6P receptor that span Golgi
-serve as binding site for adaptor proteins which then bind to clathrin
(various GTP binding and adaptor proteins play key role in assembly of vesicles for different destinations) |
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Term
| Describe Vesicle fusion: SNAREs |
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Definition
-vesicle fusion is mediated by interactions between pairs of transmembrane proteins called SNAREs
-vesicle and target = v/t SNAREs
-SNARE-SNARE binding provides energy to bring two bilayers close enough to destabilize (dispel water) them to fuse |
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Term
| Describe Vesicle fusion: Rab proteins |
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Definition
-GTP binding proteins that play a role in docking of transport vesicles
-Rab proteins mark different organelles and transport vesicles, so they establish specificity in vesicular transport
-carried through cytosol in GDP bound form
-activated by GEF that is bound to membrane (converts to GTP)
-Rab are only active with GTP when at membrane |
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Term
| Describe Vesicle fusion: Process |
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Definition
1. Rab/GTP on transport vesicle interacts with effector proteins and v-SNAREs to assemble pre-fusion complex
2. Different Rab on membrane organizes effector proteins and t-SNAREs
3. effector proteins link membranes by tethering
-Rab/GTP is converted to GDP
4. v and t SNAREs contact and bring membranes into nearly direct contact and membranes fuse!
5. NSF/SNAP complex disassembles SNAREs
-requires ATP |
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Term
| Describe Vesicle fusion: Exocytosis |
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Definition
-fusion of vesicle to plasma membrane resulting in secretion of contents
-this occurs at protein complexes called exocysts |
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Term
| Describe Vesicle fusion: Controlled release |
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Definition
Axon: release is controlled by electrical potential
-depolarization comes down axon and allows Ca2+ to bind to vesicle/effector
-allows fusion and contents are released |
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Term
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Definition
-membrane enclosed organelles that contain enzymes to break down biological polymers
-digestive system of cell
-vary in size and shape depending on contents
-acid hydrolases
ex. nucleases, phosphatases, lipases, glycosylases
-mutations can lead to lysosome storage diseases |
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Term
| Describe lysosomal environment |
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Definition
-enzymes only active at pH=5 and cytoplasm in 7.2
-provides protection in case they break open
-pH is maintained by proton pump that actively transports (ATP) protons into the lysosome |
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Term
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Definition
-vesicles that represent intersection between secretory pathway and endocytic pathway
Early: receive endocytic vesicles from plasma membrane
-separate molecules targeted for recycling (back to membrane) from those destined for degradation in lysosomes
Late: receive lysosomal enzymes from Golgi and fuse or mature into lysosome |
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Term
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Definition
-specialized cells take up and degrade large particles
-particles are taken up in phagocytic vacuoles (phagosomes) which fuse with lysosomes to form phagolysosomes
-similar to autophagy
*organelles |
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