lecture 4

Question 1

where do cytosolic ribosome-made proteins end up

Answer 1

• remain in cytosol
• or are TARGETED to intracellular organelles
• e..g ER, mitochondria, chloroplasts, peroxisomes, nucleus
  ** must have specific signal sequence

Question 2

where do ER ribosome-made proteins end up

Answer 2

• SORTED to PM, Golgi, lysosomes or can remain in ER

Question 3

what are the two major protein-sorting pathways

Answer 3

consecretory and secretory

Question 4

nonsecretory pathway

Answer 4

• proteins that go to lysosome

Question 5

secretory pathway

Answer 5

• proteins that go to secretory vesicle

Question 6

ER

Answer 6

• uninterrupted membranous tubules and vesicles separated from cytoplasm
• RER has ribosomes on the tubules

Question 7

what are the cisterna

Answer 7

• sacs/tubules that make up the ER?, inner parts of the tubules

Question 8

where does the eR extend from

Answer 8

nucleus

Question 9

what does the TEM allow us to see, that light microscopy would not

Answer 9

• the ribosomes

Question 10

in what direction do ER ribosomes face

Answer 10

• towards the cytosol

Question 11

what did experiment 1 with the detergent show?

Answer 11

• that secreted proteins must first go through the ER

Question 12

describe the experiment that showed that secreted proteins must first go through the ER

Answer 12

• we know that antibodies are examples of proteins that are secreted outside of the cell
• so they homogenized cells that expressed a certain antibody
• homogenization disrupts the ER, and breaks it apart
• ER re-forms as vesicles called microsomes
• microsome splits into two
• 1/2 is treated with non ionic detergent first, and then protease is added to chew up the immunoglobin
• 1/2 is treated only with protease
• after running SDS page, second example still has protein present
• adding detergent allowed the protease to get in and chew up the immunoglobulin, otherwise cannot passed microsomal membrane.

Question 13

how do we know that translocation and translation occur simultaneously?

Answer 13

• experiment involving in vitro translation
  1) all the pieces of the translation apparatus were put in a tube with a specific mRNA. allow the protein to be synthesized for half an hour, and then add the microsomes. when these proteins were analyzed by SDS page, it was observed that none of the protein was found in the lumen of the microsomes.
  2) mixing everything together, mRNA, in vitro translation concoction, microsomes. secreted protein was now found in the lumen of the microsome, and had undergone furtherr processing to cleave off a certain piece
  ** this shows that translation and translocation occur simultaneously

Question 14

translocation of proteins

Answer 14

• ## movement of protein from outside into lumen of ER

Question 15

nascent proteins

Answer 15

• newly initiated polypeptide

Question 16

where is signal sequence located

Answer 16

amino terminal?

Question 17

where is the SRP receptor embedded

Answer 17

ER membrane

Question 18

SR[

Answer 18

signal recognition particle

Question 19

translocon

Answer 19

protein channel

Question 20

cleavage site

Answer 20

• where signal sequence is cut by a signal peptidase

Question 21

what protein modifications take place in the ER

Answer 21

• specific proteolytic cleavage (signal peptidase cleaving off the signal sequence)
• glycosylation (addition of sugar groups)
• formation of covalent disulfied bonds
• folding of polypeptide chains with the help of helper proteins

Question 22

glycosylation in the ER

Answer 22

• there is a complex sugar structure made up of 14 residues
• it is attached to the dolichol on the membrane, linking the sugar structure to the membrane
• proteins that start to emerge in the lumen of the ER will be recognized by portions called oligosaccharyl transferases, which will transfer the oligosaccharide onto an asparginine residue of the polypeptide.
• the glycosides can work to modify N glycoln which is covalently attached to the protein at the Asp residues.

Question 23

what is the purpose of glycosylation

Answer 23

• it has functions in protein folding, conferring protein stability, cell adhesion, e.g. EC matrix proteins are sticky and glycosylation and sugar residues are responsible for this stickiness

Question 24

what proteins contribute to proper folding of proteins in the ER (after glycosylatio> )

Answer 24

BiP, lectins calnexin and calreticulin, and PDI
- they present the midsolfing or aggregation of nascent proteins

Question 25

when can proteins be transported fom RER to GOlgi

Answer 25

only when they are properly folded

Question 26

why are helper proteins important for protein folding

Answer 26

• cotranslation into the ER lumen is that it can make the cell vulnerable to misfiolding or aggregation
• hydrophobic amino acids will try to stick to each other, so chaptern proteins will prevent them from misfolding

Question 27

BiP

Answer 27

• ER chaperon prtotein that binds directly to emerging polypeptide that may start to misfold, usually on hydrophobic stretches.

Question 28

oligosaccharyl transferase function for protein folding in the ER

Answer 28

• might receive a signal to stick on a branched sugar to maybe a hydrophobic stretch of amino acids or part of the peptide that is starting to misfiled.
• this can allow it to be recognized by other chaperon proteins (calnexina dn calreticulin)

Question 29

where is calretifuclin found

Answer 29

• floating in the eR

Question 30

where is calnexin found

Answer 30

• in the ER membrane

Question 31

PDI (another helper protein)

Answer 31

• forms transient disulphide bonds to hold on tot eh sulfur atoms to help form and properly form those sulfur bonds for structural stability.

Question 32

targeting

Answer 32

directs proteins to the right destination (organelles), happens during or after synthesis

Question 33

sorting

Answer 33

directs proteins to the lysosomal and secretory pathways (ER —> Golgi, lysosomes)

Question 34

stringing the concepts together

Answer 34

• identify cellular features by microscopy isolate and homogenize to free the organelles
• use sucrose desinity-gradient centrifugation of homogenate to allow for isolation of microsomes and ribosoomes
• use SDS page to identify newly translated proteins

Question 35

what is the difference between the two types of protein synthesis (microsomes present vs not present)

Answer 35

• when the microsomes are present, it results in the creation of a mature protein that has a signal sequence
• when the microsomes are not present, there is not incorporation into microsomes, and therefore no removal of signal sequences