Intracellular compartments and Protein Sorting

Question 1

How much of cell volume does the nucleus make up

Answer 1

10%

Question 2

What membrane is the ER continous with?

Answer 2

The nuclear membrane

Question 3

What makes up the sorting and exocytotic network

Answer 3

ER, Golgi, and secretory vessicles

Question 4

What makes up the endocytic network?

Answer 4

Plasma membrane endosomes and lysosomes

Question 5

Where do peroxisomes derive from

Answer 5

From the ER and they are oxidizing vessicles

Question 6

mitochondria

Answer 6

endosymbiot ATP generating organelles

Question 7

What percent of total cell membrane composition is the plasma membrane?

Answer 7

2%, the rest is internal membranes

Question 8

Describe gated transport

Answer 8

Transport through nuclear pores

Question 9

Describe transmembrane transport

Answer 9

Translocation across a limiting lipid bilayer.

Question 10

Describe vesiculat transport

Answer 10

transport withing lipid bilayer enclosed organelles

Question 11

Describe an ER signal sequence

Answer 11

has a hydrophobic center

Question 12

Describe mitochondrial signal sequence

Answer 12

aphipathic alpha helices

Question 13

Describe a nuclear import signal

Answer 13

very basic amino acids (Lys, Arg)

Question 14

Describe a peroxisomal signal

Answer 14

SKL-COOH

Question 15

Describe a nuclear export signal

Answer 15

Leucine rich

Question 16

Describe the structure of a nuclear pore complex

Answer 16

8 proteins make up the edge (basket) of the nuclear pore. Fibrils extend cytosolically and nuclearly. The fibrils attach themselves to cargo and the pore is permeable to proteins 60kDA or less.

Question 17

What are the concentrations of RAN-GTP in the cytosol vs the nucleus

Answer 17

Low RAN-GTP in the cytosol and high in the nucleus

Question 18

What do GEF and GAP do?

Answer 18

GAP- GTPase activating protein. hydrolyzes GTP to GDP converting RanGTP to RanGDP

GEF- Guanine nucleotide exchange factor. Exchanges GDP for GTP. Catalyzes RanGTP formation in the nucleus

Question 19

Describe the Ran GTPase cycle in relationship to nuclear export

Answer 19

In order for a protein to leave the nucleus it has to have a nucleus export signal and bind to an export protein. The cargo and the export protein bind together along with GTP. This complex binds to the fibrils leaves the nucleus through the nuclear pores. In the cytosol RanGAP hydrolyzes the GTP to GDP releasing the cargo into the cytoplasm. The GDP and expotin go back to the nucleus where GDP is exchanged for GTP bt GEF

Question 20

Describe the RanGTPase cycle in relationship to nuclear import.

Answer 20

Importin attaches to cargo with a lysine rich nuclear import signal. The complex binds to the nuclear pore and goes into the nucleus. RanGTP binds to the complex and stimulates disassociation of the cargo. GTP still bound to importin travels back to the cytosol where RanBP1 and RanGAP act on it to hydrolyze GTP disassociating it from imortin. GDP travels back to the nucleus where GEF acts on it to make GTP again and the cycle continues

Question 21

Describe the regulation of NFkB

Answer 21

NFkB exists in the cytosol bound to its inhibitor I-kB. When I-kB is phosphorylated it disassociates from NFkB revealing its nuclear import signal. NFkB gets taken into the nucleus and activates genes

Question 22

How do nuclear proteins get back to the nucleus after mitosis

Answer 22

After the nucleus breaks down nuclear proteins have nuclear localization signals still bound to them. After mitosis the cell divides and the proteins are divided, the proteins go where they belong in the cell based on their attached signals

Question 23

How many membranes and compartments does the mitochdonrion have?

Answer 23

2 membranes

4 compartments

• outer membrane
• inner membrane
• intermembrane space
• mitochondrial matrix

Question 24

What encodes mitochondrial proteins

Answer 24

Majority of mitochondrial proteins are encoded by the nuclear genome, even though mitochondria have their own DNA. Mitochondrial proteins recieve a mitochondrial localization signal so they get shuttled to the mitochondria (interdependency)

Question 25

Describe the mitochondrial signal protein and how it binds to the importin.

Answer 25

The mitochondrial signal protein is a an N-terminal amphipathic alpha helix with a + charged face. The import protein have a - charged divet that it grabs onto the + charge to transport the mitochondrial protein

Question 26

Describ the steps of protein import into the mitochondria

Answer 26

Protein with a mitochondrial localization signal binds to TOM (translocase of outer membrane).Cytosolic Hsp70 unfolds the protein (using ATP) and slides it through the aqeous pore. It then binds to TIM (Translocase of inner membrane) to get inside the mitchondrial matrix. The electrochemical potential of the inner membrane is dissipated to electrophorese the unfolded protein into the matrix space. When the protein is inside the matrix mitochondrial Hsp70 uses ATP to pull the protein through the pore. Once in the signal peptide is cleaved.

Question 27

Once inside the mitochondrial matrix what protiens help the unfolded protein to fold

Answer 27

Hsp70 binds to the unfolded protein domains and protects them while the rest of the protein folds up. Once folded they will dosassociate

Hsp60(GroES) is a barell shape and encloses the unfolded protein in a microenvironment so that it can fold.

Both processes are ATP dependent.

Question 28

Functions of Peroxisomes

Answer 28

Oxidation of long chain fatty acids

Oxidation of branched chain fatty acids

Oxidation of cholesterol to bile in the liver

synthesis of plasmalogens (membrane lipids found in myelin)

Oxidation (detoxification) of some metabolic intermediates and foreign substances for elimination (flavoprotein oxidases and catalase)

Decomposition of hydrogen peroxide

beta-oxidation of fats to make acetyl Coa

Question 29

Which organelles do most of the oxidation in cells

Answer 29

mitochondria, but peroxisomes isolate oxidtion reactions

Question 30

What is the key redox reaction in peroxisomes?

Answer 30

Catalase

H2O2 + H2O2 ⇒ 2H2O + O2

S’H2 + H2O2 ⇒ S’ H2O

Question 31

How do peroxisomes arise?

Answer 31

come from the ER by a budding process. They are expanded by acquiring new membrane from the ER and then dividing by fission

Question 32

How do peroxisomal proteins get into the peroxisome?

Answer 32

They are imported from the cytsol after the peroxisome is formed. All proteins go into the peroxisome post translationally through signals on their C terminus

Question 33

Function of smoothe ER

Answer 33

release and reuptake of calcium and contains enzymes for detox

lipid biogenesis

Question 34

Describe the function of the rough ER

Question 35

Function of the transitional ER

Answer 35

Vessicles and ER cargo bud off here.

Question 36

What occurs when a protein has a an ER localization signal.

Answer 36

The ribosome pauses translation and localizes with ER. So as the protein is synthesized it is pushed through an aqeous pore into the ER. Energy for this comes GTP hydrolysis.

Question 37

What is the ER lumen topologicaly equivalent to

Answer 37

The outside of the cell.

Question 38

Steps in co-translational transport

Answer 38

A protein being translated has an ER signal peptide, which is recognized by a signal recognition protein. The ribosome goes to the ER pore Sec61, translation continues forcing the protein through the pore. When translation completes the protein is in the ER lumen, the signal is cleaved by a signal peptidase, and the ribosome disassociates.

Question 39

How are transmembrane proteins localized in the membrane

Answer 39

Membrane proteins have a stop transfer sequences. The stop transfer sequences is a stretch og hydrophobic residues flanked by charged residues (the se are the start and stop signals) When a ribosome reaches a stop transfer it will finish tranlation cytosolically. The stop sequence will stay in the Sec61 and will come out into the membrane when the Sec61 is pried open.

Question 40

How does a transmembrane proteins get either its amino or carboxy end to to face the cytosol?

Answer 40

The orientation of the peptide within the ER Sec61 channel depends on the amino acid sequences that flank the hydrophobic core. Whichever end of the hydrophobic core is more positive will face the cytosol

Question 41

How do you get a multipass transmembrane protein?

Answer 41

Multiple start and stop sequences. start-start-stop-start-stop. hydrophibic sequences alternate between them.

Question 42

Describe the attachment of mannose rich oligosaccharide units to aspargine residues in N-linked glycoproteins

Answer 42

Oligotransferase transfers sugar to the asparagine residues in the sequence Asn-X-Ser/Thr. The sugar is atached to the nitrogen in the Asn. Glucosidases will act to trim the sugar, and finaly mannosidase will trim it into a mannose. It can now go to the Golgi

Question 43

What is the function of N-glycan structures?

Answer 43

1) To control ER folding
2) To protect the underlying protein from proteases or antibodies or other attacks.
3) To add hydrodynamic volume to the protein.
4) To operate as part of a sorting signal
5) To provid inter-cellular communication via lectins

Question 44

Identify the lipid that participates in the assembly and transfer of the high mannose-oligosacharride chain in the N-linked glycoproteins.

Answer 44

Dolichol lipid

Question 45

Describe the unfolded protein response

Answer 45

1) Misfolded proteins in the ER signal the need for more chaperone proteins by activating a transmembrane kinase
2) Activated kinase turns into a endoribonuclease
3) Endoribonuclease cuts specific RNA molecules at 2 positions removing an intron
4) two exons are ligated to form an active mRNA
5) mRNA is translated to make a gene regulatory protein
6) Gene regulatory protein enters nucleus and activates genes encoding ER chaperons
7) Chaperones are made in the ER where they help to fold proteins