Unit 4

Question 1

Where does translation begin? Is there an order to how subunits are attached?

Answer 1

the cytosol. Random order - any large subunits can attach to any small subunits - mix and match

Question 2

Polyribosomes

Answer 2

Multiple ribosomes work simultaneously on a single mRNA. As soon as a ribosome has translated enough of the nucleotide sequence to move out of the way, a new ribosome binds to the 5’ end of the mRNA

Question 3

When are proteins folded?

Answer 3

During translation

Question 4

The endomembrane system

Answer 4

Membrane compartments involved in synthesis/processing and movement of proteins, lipids, and carbohydrates

Question 5

Requirements for protein import into an organelle

Answer 5

1) A specific signal sequence in the protein primary sequence
2) A specific protein receptor on the organelle of interest

NOTE: proteins do not have to be fully translated or fully folded

Question 6

Where do proteins enter the endomembrane system?

Answer 6

They enter the ER and they never return to the cytosol. They either stay in the ER or continue to move to other organelles of the endomembrane system

Question 7

Are rER and sER separate and discrete membranes?

Answer 7

No - they are continuous and interconnected. Lumen and membrane of rER and sER are connected because they are one organelle. The sER and rER ratio depends for every cell

Question 8

Import to the ER signal sequence

Answer 8

Consists of many nonpolar amino acids. 20-27 AAs at the N-terminus serve as the signal sequence (start transfer sequence). It contains a segment of approx. 10< hydrophobic AAs

Determines location and orientation in the ER

Question 9

When is the signal for entry into the ER recognized?

Answer 9

Before translation of the protein is complete

Question 10

What are the differences between ER membrane-bound and free ribosomes?

Answer 10

1) they differ only in the proteins they are making at a particular time
2) they differ in their sequence, if they contain a signal sequence that targets them to the ER or not

Otherwise, they are structurally and functionally identical

Question 11

Post-translational trafficking of proteins

Answer 11

Ribosomes remain “free” in the cytosol

Completed polypeptide goes to its functional destination depending on its sorting signal

Question 12

Co-translational trafficking of protiens (protein is not fully finished)

Answer 12

Ribosomes attach to ER membrane

Protein is threaded through ER membrane as it’s being translated

Proteins either stay in the ER, or continue to other compartments of the endomembrane system

Question 13

Name 4 examples of proteins that would you expect to be targeted to the ER

Answer 13

1) Soluble proteins destined for secretion
2) Lysosome resident proteins
3) Enzymes required for protein glycosylation
4) Plasma membrane transmembrane proteins

Question 14

How does a protein stay in the lumen of the ER?

Answer 14

The protein should have a retention signal, called the KDEL sequence

Question 15

Targeting sequence in the primary sequence function

Answer 15

Directs the protein to a specific organelle

Must be present for protein to leave the cytosol compartment

Question 16

2 targeting requirements to the ER

Answer 16

1) Signal sequence encoded within the protein

2) Receptor that recognizes and binds the signal (SRP - signal recognition particle)

Question 17

Signal recognition particle (SRP) as the ER receptor

Answer 17

SRP recognizes the ER signal sequence (start transfer seq) and directs the protein to the ER

Question 18

Co-translational transport across the ER membrane steps

Answer 18

1) SRP binds to the exposed ER signal seq and to the ribosome (and slows down protein synthesis)
2) SRP-ribosome complex binds to SRP receptor in the ER membrane. SRP is released and ribosome is passed on to the protein translocation channel
3) ER signal seq binds the protein translocation channel and opens it, and polypeptide chain gets threaded through the channel across the lipid bilayer

Question 19

What does the primary seq of the proteins determine according to the ER

Answer 19

Location in the ER and orientation in the ER membrane

Question 20

Start transfer seq (N-terminal signal seq or N-terminal start transfer seq) and internal start transfer seq

Answer 20

Initiates transfer of protein across membrane. It is cleaved off

Initiates transfer of protein across membrane. It is a membrane-crossing domain and not cleaved off

Question 21

Stop transfer seq

Answer 21

Stops transfer of protein across membrane. It is a membrane-crossing domain

Question 22

Where will a protein with only an N-terminal ER signal seq ultimately end up?

Answer 22

Outside the cell - once it is in the ER, it cannot go back to the cytosol. Since it has a signal seq, it will go to the ER but will not stay since it does not have a retention signal

Question 23

Where will a protein with an N-terminal ER signal seq and an NLS ultimately end up?

Answer 23

Outside the cell - the NLS might not be translated. The protein needs to be fully made before it goes to the nucleus. ER doesn’t need a fully-formed protein

Question 24

Orientation of transfer sequences

Answer 24

First transfer seq is the “start” transfer seq and allows the polypeptide to feed into the ER after the seq. If it is at the N-terminus, it will be cleaved off after protein synthesis

Stop transfer seq follows start. causes polypeptide to stop entering the ER after stop seq

Stop and start seq alternate when several are present

Question 25

Function of ER regarding protein processing

Answer 25

Protein folding Covalent modification Disulfide bond formation Glycosylation

Question 26

Protein folding in the ER

Answer 26

Proteins fold via hydrophobic interactions. When there are many polypeptides being synthesized and co-translated, they can form unwanted interactions with each other, which can result in new polypeptides clumping. In the ER, chaperone proteins prevent this by "holding on" to the proteins until they fold properly. They also prevent misfolded proteins/partially assembled proteins from leaving the ER

Question 27

Chaperone proteins production in the ER

Answer 27

Misfolded proteins in the ER lumen initiate the production of chaperone proteins. Feedback loop.

Question 28

What happens to misfolded proteins?

Answer 28

They are tagged with ubiquitin and sent back to the cytosol and degraded by to proteasome. AAs are recycled

Question 29

After the translation on ribosomes in the cytosolic compartment, where are proteins processed?

Answer 29

Either in the cytosol or in the ER/Golgi system

Question 30

Glycosylation - oligosaccharide units

Answer 30

Begins in the ER. An oligosaccharide "tree" is added to dolichol (a phospholipid) on the cytosolic face of the ER membrane. Then a flippase enzyme flips dolichol in the membrane so the oligosaccharide tree is in the ER lumen side. Lastly, the tree is transferred from dolichol to the growing polypeptide chain, where a transferase enzyme recognizes the Asn-X seq and links the tree to it. Glycoprotein is packaged into vesicle and sent to Golgi. Based on the protein structure, sugars are added, and this overall structure determines where it is sent

Question 31

What happens to the oligosaccharide in the Golgi

Answer 31

In the Golgi and the ER, the oligosaccharide is trimmed, then in the Golgi, new sugars are added to the oligosaccharide one at a time

Question 32

Requirements for vesicles to take the correct cargo from the donor compartment to the correct target compartment

Answer 32

Cargo getting into the correct vesicle depends on the vesicle protein coats. The vesicle getting to the correct destination depends on Rab proteins, Tethers, and Snares

Question 33

Vesicle formation

Answer 33

The cargo protein is recognized by the cargo receptor and bind in the membrane. The adaptor proteins recognize and bind to the cytosolic portion of the receptor and the coat proteins bind to the adaptor proteins. These coat proteins bend the membrane into vesicles and the vesicle pinches off from the donor membrane

Question 34

Steps of a vesicle budding off

Answer 34

The process is regulated by GTP-binding protein. When it is bound the GTP, it is active and allows adaptors and coat proteins to bind. When GTP is hydrolyzed to GDP, it becomes inactive and GTPases (binds GTP and helps assemble membrane coat), coat proteins, and adaptors dissociate from vesicle

Question 35

What would happen if the dynamin protein is defective?

Answer 35

The dynamin protein acts as a pair of molecular scissors and helps newly formed vesicle proteins to bud off from the plasma membrane. If it is defective, the vesicles cannot bud off

Question 36

Do all newly forming vesicles require adaptin, clathrin, and dynamin on the cytosolic face of the membrane?

Answer 36

No, there are different adaptors and coat proteins involved in vesicle formation in the cytosol, such as clathrin, COPI, and COPII

Question 37

Clathrin-coated vesicles

Answer 37

Involved in PM to endosome traffic (endocytosis) and Golgi to lysosome (via endosome). Dynamin is required for budding

Question 38

COPI-coated vesicles

Answer 38

Involved in Golgi to ER traffic (retrieval). Dynamin not requried

Question 39

COPII-coated vesicles

Answer 39

Involved in ER to Golgi traffic (forward). Dynamin not required

Question 40

Two proteins that direct vesicles to the right place for docking

Answer 40

1) Rabs are proteins that are lipid-linked to the membrane | 2) Tethers are proteins that bind to the Rab on the vesicle and pull them into the PM, which eventually fuse

Question 41

2 proteins that mediate vesicle fusion

Answer 41

1) v-SNAREs are on vesicle membranes and are made up of one protein 2) t-SNAREs are on the target membrane and are made up of two or more proteins

Question 42

Difference between Golgi in mammalian and plant cells

Answer 42

In mammalian cells, the Golgi is a singular perinuclear stack, while plant cells have small stacks on tracks that move around the cell (b/c of cytoskeleton)

Question 43

Cisternae of Golgi

Answer 43

Each one contains different enzymes that modify the proteins that pass through it, via adding/removing sugars to the oligosaccharide (covalent bonds)

Question 44

Protein sorting in Golgi

Answer 44

Proteins in the cis face can either move forward, stay in cis Golgi, or return to the ER via retention signal Proteins in the trans face are sorted via their oligosaccharide signal as regulated secretion, constitutive secretion, or via the lysosomal pathway

Question 45

2 major proteins in the Golgi

Answer 45

Cargo proteins - passes through Golgi to move to other destinations in the endomembrane system (ex. proteins to be secreted, sent to lysosome, etc.) Resident proteins - those that function in the Golgi (ex. glucosyl transferase, etc.)

Question 46

Vesicle transport model

Answer 46

Cargo is carried forward from cis to trans in vesicles and resident proteins stay in place in the cisternae (one direction)

Question 47

Cisternal maturation

Answer 47

Vesicles carrying the cargo fuse to form the cis-cisternae, where the cargo remains in the cisternae. When a new cis-cisternae forms, the old one "matures" to the medial-cisternae, etc. Resident proteins must move backwards (trans to cis) as cisternae moves forwards (cis to trans)

Question 48

Exocytosis: Constitutive secretion (default secretory pathway)

Answer 48

Occurs in all cells and happens continuously. No env signals are needed for vesicle fusion with the PM It supplies the PM with newly made lipids and proteins, allows it to expand before cell division. Also lets secreted proteins to be incorporated into the cellular matrix, and allows them to nourish or signal other cells

Question 49

Exocytosis: Regulated secretion

Answer 49

Only occurs in specialized secretory cells. Proteins are stored in secretory vesicles and are only released when a signal is received (ex. digestive enzymes, insulin, etc.) It allows the proteins to be secreted rapidly and on demand

Question 50

Lumenal pH

Answer 50

As the pH increases, it initiates proteins to form aggregates and they increase in concentration and condense in secretory vesicles. These secretory vesicles lay close to the PM. This occurs to proteins in the secretory pathway

Question 51

Pulse-chase and autoradiography

Answer 51

Label one group of molecules via radio active AAs (which is the pulse) and follow where they go over time. Instrumental determination of pathways

Question 52

Green Fluorescent Protein (GFP)

Answer 52

Cargo proteins can be tagged with GFP in live cells

Question 53

Mannose-6-phosphate

Answer 53

A targeting signal and a phosphorylated sugar that is added onto lysosomal proteins. It is added during the glycosylation in the ER

Question 54

Proteins targeted to lysosomes requirements

Answer 54

1) M6P targeting signal | 2) M6P receptor

Question 55

Mannose conversion

Answer 55

Mannose is phosphorylated to M6P in the cis Golgi

Question 56

Lysosomal pathway

Answer 56

M6P signal is added to lysosomal proteins in cis Golgi and the M6P receptor in the membrane of TGN recognizes and binds to the sorting signal. The vesicle is coated in clathrin and sheds it in the cytosol to fuse with an early endosome, with a pH 6 or less env. Lysosomal enzymes dissociate from the M6P receptor in early endosome and phosphatases prevents rebinds with M6P receptor by removing lysosomal enzymes. M6P receptors are recycled back to the trans Golgi

Question 57

Endosomal compartment

Answer 57

Acts as a sorting station for proteins arrive from the TNG and cell surface. Early endosome matures into late endosome, which can turn into a lysosome, or fuse with one

Question 58

Lysosome

Answer 58

Membrane-enclosed organelle where intracellular degradation occurs. Only works at pH 5. Diverse size and shape

Question 59

Do plants have lysosomes?

Answer 59

No, plants don't have lysosomes, but their vacuoles act as lysosomes

Question 60

Can lysosomes digest their own membrane proteins and lipids?

Answer 60

No; they are resistant to their own digestive enzymes because of extensive glycosylation of proteins on the lumen side of the membrane

Question 61

Why don't lysosomal enzymes (acid hydrolases) digest the cell as soon as they are synthesized and before they reach the lysosome?

Answer 61

The enzymes need very low pH (approx. 5) to be activated. This is achieved through ATP hydrolysis to pump protons into lysosomes

Question 62

Endocytosis: Phagocytosis

Answer 62

"Cellular eating". A defense mechanism used to remove unwanted material (ex. bacteria) and target it for degradation in the lysosome. This occurs by pseudopods, which are cell extensions produced by changes in the cell's cytoskeleton, that engulf the bacteria. No coat proteins are used. The phagosome fuses with lysosomes and the material taken by phagocytosis is degraded by lysosomal enzymes

Question 63

Endocytosis: Pinocytosis

Answer 63

"Cellular drinking". Non-selective process where fluid and macromolecs from extracellular region are taken up freely. No specific receptor required. Carried out by clathrin-coated vesicles. They fuse with an early endosome and the trash goes to late endosome, then lysosome for breakdown. This is a continuous process where cells remove membrane added by exocytosis, allowing recycling of the PM

Question 64

Receptor-mediated endocytosis (RME)

Answer 64

Cells internalize parts of the PM, including some fluid and macromolecs in small vesicles from extracellular space. Clathrin is used for vesicle formation

Question 65

Adaptin

Answer 65

A bridge between the cargo and the clathrin coat

Question 66

Low Density Lipoprotein (LDL)

Answer 66

A large complex that contains lipids and cholesterol as cargo and makes them water soluble. Carries them in blood

Question 67

Compare and contrast receptor recycling in lysosomal and endocytic pathways

Answer 67

Early endosomes are sorting centers and recycle M6P receptors back to the TGN in the endocytic pathway. In the lysosomal pathway, the LDL receptors are recycled to the PM

Question 68

Early endosome

Answer 68

First to receive all endocytosed material. This is the compartment where receptors are recycled (to the TGN or PM). Can mature to late endosome when amount of material internalized by endocytosis increases

Question 69

Late endosome

Answer 69

Don't recycle material and are purely for degradation, meaning they have a lower pH than early endosomes

Question 70

What happens if there is a mutation in an adaptin that links the LDL receptor to clathrin?

Answer 70

There is no link to the receptor, therefore, the vesicle does not form and stays on the plasma membrane. This leads to high cholesterol and heart disease