MCP 1-12

Question 0

Destinations of proteins made in ER

Answer 0

• lumens or membrane of ER, Golgi, lysosomes, or endosomes
• plasma membrane
• cell exterior
• Once in ER, ind. proteins do not return to cytosol - stay in ER or move to other organelles via vesicles (requires sorting signals)
  Note - proteins made in cytosol can remain in cytosal

Question 1

Functions of the Endoplasmic Reticulum (ER)

Answer 1

• Protein synthesis/import (RER)
• Protein modification (RER)
• Protein quality control (RER)
• Lipid synthesis (SER)
• Synthesis of steroid hormones (SER)
• Detoxification of lipid soluble drugs (SER)
• Calcium storage (SER)

Question 2

Protein import into ER

Answer 2

• occurs co-translationally (only place this occurs)
• All protein synthesis begins in cytosol -> ER target signal directs ribosome to ER membrane -> synthesis continues w/ new poly. entering ER membrane or lumen
• No ER targeting sign = stays in cytosol

Question 3

Role of SRP and SRP Receptor

Answer 3

• SRP (signal-recognition particle): binds ER signal sequence as it emerges from ribo, slowing protein synthesis
• Ribo/SRP complex binds ER membrane, with SRP binding SRP receptor and ribosome binding the translocation channel
• SRP binding receptor -> SRP releases signal sequence -> synthesis resumes with protein threaded thru translocation channel
• SRP recycled to cytosol

Question 4

Import of soluble proteins into ER lumen

Answer 4

• ribo/SRP complex binds, N terminal signal sequence opens translocation channel, protein threaded through loop
• Signal peptidase cleaves off signal sequence (in lumen), protein is free and signal peptide is degraded
• ex. ER resident proteins (BiP), lyososomal proteins (nucleases), secreted proteins (insulin, growth factors)

Question 5

Import of membrane proteins into ER membrane

Answer 5

• signal sequence of these proteins located on N terminal (cleaved) or internally
• membrane spanning domains are released laterally from the translocation channel, embedded in ER membrane
• inserted in only one orientation (topology) as dictated by signal sequence

Question 6

Protein modifications in ER

Answer 6

• signal sequence cleavage (co-translational), by signal peptidase
• N-linked glyosylation (co-translational): imported proteins converted to glycoproteins (add sugars)
• hydroxylation of collagen: allows interchain H bonds to stabilize collagen
• protein folding and disulfide bond formation (help of chaperone proteins)
• assembly of multi-subunit proteins
• retention of ER resident proteins - retention sequence that is different than ER targeting signal (pass thru and retained)

Question 7

Protein Quality Control in ER

Answer 7

• exit from ER is highly selective
• Proteins folded/assembled incorrectly are kept in ER by binding to chaperone proteins
• Mistake is held until corrected or will be degraded via proteasomes
• ER controls quality of proteins shipped off to other organelles

Question 8

Unfolded Protein Response (UPR)

Answer 8

• Triggered when ER is overwhelmed by misfolded proteins (accumulate)
• UPR signals ER to expand and increase chaperones to handle unfolded proteins
• Load still too large -> apoptosis

Question 9

Membrane lipid synthesis in ER

Answer 9

Newly synthesized phospholipids are added to cytostolic side of ER membrane, redistributed by enzymes that transfer around bilayer (scramblases, flippases)

Question 10

Synthesis of Steroid Hormones

Answer 10

• smooth ER
• more smooth ER is present in cells that syn. hormones
• ex. Leydig cells (secrete testosterone) in testes have a lot of SER for testosterone synthesis

Question 11

Detoxification of lipid soluble drugs

Answer 11

• function of the SER

- Liver cells have more SER for lipid metabolism. Also detox lipid soluble drugs/alcohol

Question 12

Calcium storage in ER

Answer 12

• release of calcium mediates intracellular signaling
• ER has high concentration of calcium (1-3mM)
• in muscle, sarcoplasmic reticulum releases calcium in response to AP to induce muscle contraction

Question 13

Vesicular transport: pathways

Answer 13

1. Secretory pathway: outward transport, proteins syn. in ER are delivered to cell surface or lysosomes via Golgi
2. Endocytic pathway: inward transport, extracellular molecules taken up by PM and delivered to lysosomes (via endosomes) for degradation

Question 14

Assembly of clathrin coat

Answer 14

• protein coats have signal molecules to direct vesicle
• clathrin (major coat protein) forms a basketlike network consisting of hexagons and pentagons
• thought to drive budding by inducing curvature
• adaptins (2nd major coat protein): bind clathrin coat to vesicle membrane; interacts with clathrin and cargo receptors to select cargo molecules for transport

Question 15

Structure of clathrin

Answer 15

• 3 heavy chains
• 3 light chains
• Form 3 legged structure –> soccer ball structure

Question 16

Dynamin

Answer 16

• small monomeric GTP-binding protein
• assembles as ring around bud
• hydrolysis of GTP -> ring constricts -> vesicle pinches off
• clathrin coat quickly removed for disassembly once pinched

Question 17

Surface markers on vesicle surface

Answer 17

1. Rabs: monomeric GTPases, markers that identify membrane type, recognized by tethering proteins on target membranes
2. Tethering proteins: capture vesicles via interaction with Rabs
3. SNAREs (v on vesicles, t on target membrane): determine if vesicle brought in by tethering protein is the right match -> dock or release
   - Fusion of vesicle requires energy (water must be displaced), catalyzed by SNAREs - wrap around each other and pull memranes close enough to fuse