Chapter 9: Protein Trafficking

Question 1

1. General eukaryotic cell architecture

2. Concept of surface area to volume ratio

Answer 1

1. • divided into various membrane enclosed compartments = organelles w/I cytoplasm -> compartmentalization of metabolic processes
   • cytoplasm contains cytoskeleton which is meshwork of proteins-> structure
   • first cells thought to resemble bacteria w/ no organelles and PM serves all cell func.
2. • bacteria can get by using only PM because of their size (S:V ratio high enough to support all needed cell func.)
   • cells constrained by SA and V ratio
   • as cells increase their size, their s/v ratio decreases = reduced rates of diffusion and exchange of chem.

Question 2

1. Hypothesized steps of the endosymbiotic theory

Answer 2

1.) ancestral cell evolved endomem. components, like er and nucleus ->cell mem. invaginations
2.) This cell ate aerobic bacteria -> mitochondria but then once cell forms, that’s the ancestral heterotrophic cell.
3.) In a 2nd event, some cells (that also contain the aerobic bacteria) consumed photosynthetic bacteria -> chloroplasts (organelle) -> ancestral photosynthetic plant cell
• AC & Bacteria = symbiosis

Question 3

1. Ways how organelles are thought to have evolved?

Answer 3

1.) Endomembrane System = nucleus, ER, Golgi, endosomes, lysosomes formed by membrane invaginations
• Interior of organelles communicate alot using small vesicles
• Nucleus enclosed by 2 mem.= consistent w/ invagination
2.) Bacterial Ingestion = sym. relationships -> mitochondria and chloroplasts

Question 4

• Evidence for the endosymbiotic theory

Answer 4

1. ) New m/c arise through binary fission, cell div. prokaryotes
2. ) M/c contain own DNA similar in structure and seq. to bac
3. ) Transport proteins in m/c similar to proteins on surface of bacteria
4. ) M/c contain own ribosomes which more similar to prokaryotic ribosomes than eukaryotic

Question 5

structure/ function of following:

1. nucleus
2. nucleolus
3. nuclear lamina

Answer 5

1. • largest mb organelle, double layer mem. called NE separates nucleus/cytoplasm. protects dna from outside, command center, houses chromatin (dna and proteins can condense into chromosomes)
2. • nucleolus: dense structure of DNA w/I the nucleus where ribosomes made
3. • nuclear lamina: fibrous meshwork of intermediate filaments found on inner surface of ne. plays a role in nuc processes, transcription, chromatin organization, dna rep, cell cycle reg.

Question 6

1.

• structure of the nuclear pore in detail and how each component is involved in the overall function

Answer 6

• allows sub. to travel in/out of nucleus (mrna, ions)
• made of over 30 diff. proteins called nucleoporins -> allow channel for passage.
• contains unstructured region part of pp chain which extends in pore and creates limiting meshwork
• allows dissolved substances (nuc.) to pass but no large substances.
• nuc porins form ring of 8 subunits
• either side of NE have nuclear fibrils and cytosolic fibrils that extend to initiate transport through pore.

Question 7

1. The mechanism of mRNA export. All components, sequences, proteins involved and the function of each.

Answer 7

• large molecules like mrna/ proteins can’t just move from nuc. to cyto.
• have to shuttled through mem. by specific proteins involved in import/export.
• mrnas bind their export protein to form ribonucleoprotein (rnp)
• rnp moves through nuc. mem. and mrna is released for translation.

Question 8

• How GTPases, GEFs, and GAPs function

Answer 8

• Nuclear import and export driven by GTP hydrolysis
  Ran-GTPase: enzyme that hydrolyzes GTP (2 cc, GTP or GDP bound)
• accessory proteins convert Ran 1 form to other:
  GEF: guanine exchange factor (in nuc)
  GAP: GTPase activating protein (in cytosol)

Question 9

• The steps GTPases, GEFs, and GAPs cycle through during nuclear import/export

Answer 9

• Once I protein and cargo get into nuc. Ran-GTP binds/removes cargo from I protein -> I protein (and itself) to export -> cytosol where GAP waiting.
• In cyto, GAP converts Ran-GTP to Ran-GDP causing it to dissociate from i protein
• The I protein free to go find another cargo for import
• Ran-GDP -> nuc. where GEF waiting
• GEF causes -> Ran -> release GDP and bind GTP

Question 10

Differentiate between translation of cytosolic vs membrane proteins as well as free vs membrane bound ribosomes (can you follow both GP and EpR from transcription through arrival at their site of function?)

Answer 10

• mrna transcribed from jerrys epr and gp gene exported from nuc. as rnp then moved to ribo for translation -> proteins.
• Because glycogen breakdown takes place in cyto, gp is a cytosolic protein and will be synthesized on free ribosomes.
• mem. bound proteins translated by attached ribosomes on rough er, allows them to enter er and packaged/ trafficked to right organelle
• epr is mem bound receptor for epi and will be translated into rough er. after gets translated into er mem. pinches off from mem in vesicle. vesicle moves to cis face of the golgi, fusion of vesicle and of golgi mem, moves through golgi, medial region receptor protein could be changed, then goes to trans side where its pinched off in a vesicle then vesicle moves to pm, fuses with that mem. and presents on surface.

Question 11

1. Understand sorting signals and signal sequences

2. how they affect protein fate

Answer 11

1. • cell constantly making proteins. Need to be properly directed to correct location in cell.
     - some org. get proteins directly from cytosol or ER
     - fate is dependent on aa. seq. which could contain a sorting signal -> protein to correct location.
     - no sorting signal? protein remains free in cytosol.
     - typical sorting signal for protein is a seq. that’s 15-60 aa long called signal sequence.
2. • usually removed after arrival but not always, shown that deletion or swapping of signal sequences changes fate of proteins.

Question 12

How soluble/secreted proteins are translated into the ER (SRP, ER signal sequence, etc.).

Answer 12

1. • Mrna, ribosome, pp complex -> targeted to er by er signal sequence (part of growing pp)
   • when signal peptide exits ribosome srp binds it-> srp binds to an srp receptor in er mem.
   • srp receptor passes signal seq. to translocator in er mem.
   • translocator= protein complex provides channel for growing pp
   • once bound by translocator the er signal seq. opens channel and remains bound to channel.
   • as translocation cont. the growing pp pushed into er lumen, called translocation.
   • signal seq. is cleaved off by a signal peptidase and the protein is released into er lumen
   • soluble doesn’t have stop transfer sequence.

Question 13

how membrane bound proteins are translated into the ER (SRP, ER signal sequence, etc.).

Answer 13

1. SP
   - Start transfer seq. at beginning. first 15 aa, going to get cleaved off. contains stop transfer seq. farther down from signal peptide, signals the translocator to release growing pp chain sideways into er lipid bilayer
   - hydrophobic stop transfer seq. forms an alpha helix in mem. to anchor mem bound protein there.
   - ribosome finishes translation, but everything translated after stop transfer seq. is on cytosolic.
   - the signal peptidase cleaves off the signal peptide so end protein is covalently attached to membrane at the specific place on er membrane.
2. MP
   - multi pass transmem. proteins, like epr pass back and forth through mem. multiple times.
   - these proteins have integral signal sequence called start transfer seq. that not removed.
   - protein is not brought to er until start transfer seq. reached
   - stop transfer seq. are still present.
   - the combination of multiple start stop transfers give rise to more passes back and forth through mem.

Question 14

• How stop and start transfer sequences work in the translation of BOTH single-pass AND multi-pass membrane bound proteins

Answer 14

• single pass: one stop transfer, one start transfer sequence
• multi pass: start transfer at internal site and stop transfer is also at internal site. The combination of multiple start/stop transfers give rise to more passes back and forth through the membrane
• epinephrine: crosses membrane 7 times, 4 start transfer sequences and 3 stop transfer sequences

Question 15

1. The detailed mechanism of BOTH vesicle docking

2. fusing with the target membrane

Answer 15

1.

• when vesicle buds from mem. has to find correct destination to deliver contents.
• each of transport vesicles have molecular markers that’s complementary to target mem.
• once to target mem. identification depends on a class of GTPases called Rab. Bound to surface of vesicle.
• Rab takes each specific vesicle -> target location, specific rab proteins are recognized by tethering proteins on target mem. (combo of Rabs and tethering proteins provide specificity)
• additional recognition is provided by family of mem proteins called SNAREs
• once tethering protein captures vesicle, complementary snares on both vesicle/target, interact well to lock vesicle in place.
• v snares: vesicular snares
• t snares: target mem. snares

2.
v-SNAREs & t-SNAREs are responsible for catalyzing mem. fusion
• In + to delivery, fusion also introduces vesicle mem. to that of the target location (lipid intermixing)
• During fusion, the two mem. must come w/I 1.5 nm of each other which requires displacement of h2o from the hydrophilic mem surface.
• This energetically unfavorable and requires energy
• This energy barrier is breached by SNAREs that act as winch, wrapping around each other more and more tightly bringing the 2 mem. close enough for fusion

Question 16

1, The overall structure and regions of the Golgi

2. The role they play in transport

Answer 16

1. • Golgi Apparatus - Distribution center of the cell: proteins are packaged in mem-bound vesicles and sent to correct location
   • Proteins/ lipids also modified here (ex: attachment of polysaccharide side groups)
   • Folded into flattened discs called cisternae, w/ 3 main regions: (cis, medial & trans)
   • Other mem. bound organelles (lysosomes) are made here as well (pinch off as large membrane bound vesicles – budding)
2. • Vesicles from ER fuse to the cis-face of the Golgi apparatus
   • Proteins travel through the cisternae to the trans-face of the complex, where they are released in a secretory vesicle for their specific destination.

Question 17

1. Differentiate between the different exocytic pathways

2. the purpose/function of secretory vesicles (specifically the role they play in storing secreted proteins/hormones)

Answer 17

DEF: A steady stream of vesicles leave trans Golgi network & fuse w/ plasma membrane (exocytosis)
1.
• Constitutive exocytosis pathway – constantly working in ALL cells to replenish mem. lipids and proteins. Carries soluble proteins to pm for secretion (release from the cell)
• Regulated exocytosis pathway – functions ONLY in specialized cells involved in producing big amount of a specific product (Remember release of epi from adrenal cells??)
- These big amounts of product are stored in secretory vesicles in cell’s cytoplasm for later use
- Proteins secreted by this pathway have special properties -> aggregate in ionic conditions -> in trans Golgi (Acidic, high Ca2+)
- Allows them to be stored in higher conc. and when secreted ->diffuse.