Lecture 7 - Exam 3: The Endoplasmic Reticulum

Question 1

What is the In Situ Hybridization?

Answer 1

A technique that allows the detection and localization of viral nucleic acids (DNA or RNA) in tissue sections using labelled nucleic acid probes with complementary ANTISENSE sequences to the target viral nucleic acid.
Tells us when and where mRNA is transcribed, not how much!!*

Process:
Make an antisense RNA complementary to a region of a gene. We add a molecule called DIG that binds to the base U. There will be a hybridization of the antisense and sense strand of the sample. We add an Anti-DIG antibody, an alkaline phosphatase, and a NBT/BCIP that can be any color.

Question 2

If we have an image of a nucleus and the nucleolus is lit up after the in situ hybridization, what does that tell us?

Answer 2

That we have found rRNA molecules location.

Question 3

We have an electron micrograph of rough ER in rat liver cells. Ribosomes are attached to the…?

Answer 3

Cytosolic face of the ER membrane

Question 4

We have an electron micrograph of smooth ER in Leydig cells of the testis, which are active in…?

Answer 4

Steroid hormone synthesis.

Question 5

Where do proteins go during the process of sorting secreted proteins?

Answer 5

ER -> Golgi -> Secretory Vesicles -> Cell exterior

Question 6

Are free and membrane-bound ribosomes functionally distinguishable?

Answer 6

No

Question 7

Secretory proteins are targeted to the ER by..?

Answer 7

A signal sequence at their amino (N) terminus, which is removed during incorporation of the growing polypeptide chain into the ER.

Question 8

Describe signal sequences.

Answer 8

Usually ~ 10-40 amino acids and include a stretch of 7-12 hydrophobic residues.
They are usually located at the amino terminus of the polypeptide chain… but NOT always.

Question 9

What are the highlighted polar and nonpolar side chain amino acids?

Answer 9

Nonpolar: Cysteine (Cys) C
Polar: Serine (Ser) S, Threonine (Thr) T, Tyrosine (Tyr) Y

Question 10

What are the amino acids with basic (charged) groups and acidic side chains?

Answer 10

Basic: Lysine (Lys) K, Arginine (Arg) R, and Histidine (His) H
Acidic: Aspartic acid (Asp) D, Glutamic acid (Glu) E

Question 11

Who discovered signal sequences and the signal hypothesis?

Answer 11

Gunther Blobel

Question 12

Besides secretory proteins being targeted to the ER by a signal sequence at their amino (N) terminus, what is another way that secreted proteins can enter the ER?

Answer 12

Posttranslational translocation (proteins being translocated to the ER after translation). Rare in animal cells.

Question 13

What is the process of co-translational targeting of secretory** proteins to the ER? (posttranslational translocation)

Answer 13

1) SRP binds to ribosome and signal sequence on N terminus.
2) The SRP associated with the signal sequence associates with the SRP receptor and closed translocon (the ribosome’s hydrophobic R groups and the translocons hydrophobic R groups are attracted). The SRP will then dissociate and leave via GTP (hydrolysis?)
3) The signal sequence enters a membrane channel. The insertion of the signal sequences opens the translocon by moving a plug away from the channel.
4) The growing polypeptide chain enters the open translocon and signal peptidase.
5) The signal sequence detaches in the translocon and the polypeptide chain is released in the ER lumen.

Question 14

Yeast and mammal translocons are complexes of…?

Answer 14

Three transmembrane proteins called Sec61 proteins.
Also a pore ring in the center.

Question 14

Proteins that are destined for incorporation into membranes are initially inserted into the…?

Answer 14

ER membrane rather than the ER lumen

Question 15

Proteins that are destined for incorporation into membranes are initially inserted into the ER membrane rather than the ER lumen. They are transported along the __________ as membrane components rather than as ___________.

Answer 15

Secretory pathway ;
soluble proteins

Question 16

The lumen of the ER is __________ equivalent to the…?

Answer 16

topologically ;
exterior of the cell.

Question 17

In eukaryotic cells, initial sorting takes place while what is in progress?

Answer 17

Translation

Question 18

What are the different orientations of membrane proteins?

Answer 18

The proteins spanning the membrane differ in whether the carboxy (C) or amino (N) terminus is on the cytosolic side. There are also multiple membrane-spanning protein regions.

Question 19

Describe the insertion of a membrane protein with the carboxy terminus on the cytosolic side (a cleavable signal sequence).

Answer 19

Some transmembrane proteins have a normal amino terminal signal sequence. The signal is cleaved by the signal peptidase as the polypeptide chains crosses the membrane.
Translocation is inhibited by the membrane spanning domain (~25 hydrophobic amino acids). This opens the translocon and allows the hydrophobic transmembrane (TM) domain to move laterally into the membrane.
The remainder of the polypeptide chain is translocated into the ER and the cytolosolic ribosome releases and translates the rest of the polypeptide chain.

Question 20

Does the final membrane protein contain a N terminal signal sequence?

Answer 20

NO!

Question 21

Describe the insertion of membrane proteins with the N terminus on the cytosolic side (internal transmembrane sequences).

Answer 21

Other proteins are inserted into the ER by internal TM sequences that are recognized by SRP, but no cleaved by the peptidase.
These internal sequences are then brought to the translocon and the ribosome pushes the rest of amino acid chain into the lumen of the ER.
The orientation of the protein depends the amino acids immediately flanking the TM domain = positively charged amino acids stay on the cytosolic side of the translocon. This is driven by negatively charged residues near the cytosolic side of the translocon.