Unit 8: Protein Distribution + Transport, Edoplasmic Reticulum, Golgi Apparatus + Lysosomes Flashcards
1
Q
Basics
A
- Eukaryotic cells characterised by presence if membrane bound organelles in cytoplasm, in which specific activities efficiently occur
- Protein transport between different organelles is complex process
- Proteins destined for endoplasmic reticulum (ER), Golgi apparatus (AG), lysosomes (L), plasma membrane and to be secreted are synthesised in ribosomes bound to the ER membrane
- In ER, folding + processing of proteins take place
- From ER, proteins are transported in vesicles to the AG, where they are further processed + distributed for transport to lysosomes, plasma membrane or to be secreted
2
Q
Endoplasmic reticulum
8.1
A
Membrane surrounded network of tubules + sacs extending from nuclear membrane to entire cytoplasm
- Surrounded by continuous membrane
- Membrane = *50% of all cell membranes + *lumen (cistern space) = 10% of all cell volume
- *Rough ER (RER): associated with ribosomes on its outer (cytosolic) face + involved in protein synthesis + processing
- *Smooth ER (SER): does not associate with ribosomes, involved in lipid metabolism
3
Q
RER + protein secretion, Label chairing experiment
8.1
A
Pancreatic afinar cells (secrete most de novo synthesised proteins in digestive tract) labelled with radioactive aa to study intracellular pathway used by secreted proteins
- After short incubation period with radioactive aa (3min label), autoradiography revealed that de novo synthesised proteins localised in RER.
- After subsequent incubation with non-radioactive aa (chasing), proteins moved from ER to AG then, inside vesicles, to plasma membrane and to outside of cell
4
Q
- *General scheme of protein distribution in mammals
8. 1
A
- *Protein synthesised on free ribosomes: remain in cytosol or transported to nucleus, mitochondri, chloroplasts or peroxisomes
- *Proteins synthesised in RER: translocated directly into ER via *translocon (protein complex that forms channel for proteins to be introduced to ER). Can be retained within or be transported to nuclear membrane, peroxisomes or AG. From AG can be moved to endosomes, lysosomes, plasma or outer membranes via secretory vesicles
5
Q
- Labelling of proteins to target ER
8. 1
A
- Proteins translocated to ER during synthesis on membrane bound ribosomes (cotranslational translocation) or once translation completed on free ribosomes in cytoplasm (post-translational translocation)
- Mammals:most proteins enter ER via cotranslational, Yeast: both pathways used
- 1st step of cotranslational pathway is association of *ribosome-mRNA complex with ER
- ‘tag’ that determines ribosome binds to ER membrane contained in primary sequence of aa of polypeptide chain being synthesised, not sue to intrinsic ribosome properties
- Free + membrane-bound ribosomes are functionally indistinguishable. All protein synthesis begins on ribosomes free in cytosol
- Ribosomes involved in protein synthesis to be secreted labelled for targeting ER by *signal sequence located at *amino terminus of growing chain.
- Signal sequence = hydrophobic aa cleaved from polypeptide chain during transfer to ER
6
Q
Experiment that proves signal sequence
8.1
A
obtaining an *in vitro preparation of RER, for studies on direction of proteins to their appropriate locations in cell:
- When cells breakdown, ER breaks down into small vesicles (*microsomes)
- Large amount of RNA in ribosomes increases the density of rough microsomes
- Güter Babel + David Sabatini (1971) proposed that signal that directed some ribosomes to ER is at amino terminal end of chain + is deleted
7
Q
*In vitro mRNA translation experiments of secretion proteins
A
- When translation occurs in free ribosomes they gave rise to slightly larger proteins, compared to normal secreted proteins.
- When microsomes added to system, polypeptide chains were incorporated into microsomes + signal sequence was removed by proteolytic cleavage, producing a protein of the expected size
- *signal sequence contains between 15-40aa, incl. strip of 7-12 hydrophobic aa, usually at amino terminal end of chain, preceded by basic aa like Arginine (Arg)
8
Q
- Steps of cotranslational translocation of secretion proteins to the ER
8. 1
A
- As they exit ribosome, sequences recognised + attached to a signal recognition particle (SRP) made up of 6 polypeptides + a small cytoplasmic RNA.
- SRP stops translation + accompanies complex to ER membrane, where it binds to SRP receptor.
- SRP release + ribosome binds to translocon. Insertion of signal opens translocon.
- Translation resumes + signal sequences cleaved by signal peptidase.
- Translation continues, driving translocation of growing polypeptide chain across membrane.
- Polypeptide released into lumen of ER.
9
Q
Other info about cotranslational translocation of secretion proteins to ER
8.1
A
- Yeast + mammals: *translocons that cross ER membrane are complexes of 3 transmembrane proteins called *Sec61
- Insertion of signal sequence opens translocon by removing plug from translocational channel, allowing growing chain to be transferred through translocon as translation continues
- Process of protein synthesis directly produces transfer of nascent polypeptide chains through translocon to ER.
10
Q
- Post-translational translocation
8. 1
A
- Proteins translated by cytosolic ribosomes then targeted to ER (*occurs in many yeast proteins).
- Does not require SRP.
- Signal sequences recognised by *Sec62/63 complex associated with translocon in ER membrane.
- Hsp70 chaperones required to maintain polypeptide chains in their primary conformation so they can penetrate the translocon.
- Other Hsp70 chaperones within ER (called BiP) associated with Sec63 are necessary to allow chain to cross channel into ER.
- Many BiP molecules bind to chain as they undergo translocation to ER, preventing sliding backwards + propelling them through channel.
- Release of BiP molecules linked to hydrolysis of ATP.
11
Q
- How are the 2 translocation pathways activated?
8. 1
A
- Post-translational translocation activated by BiP (type of Hsp70 chaperones in ER)
- Cotranslational translocation directly driven by protein synthesis process
12
Q
- Insertion of proteins into ER membrane
8. 1
A
- Proteins destined to be secreted or reside in ER lumen, Golgi apparatus or lysosomes translocated + released into ER lumen.
- Proteins destined to be incorporated into plasma membrane or ER membrane, Golgi or lysosomes are initially *inserted into ER membrane rather than being released into ER lumen.
- From ER membrane they continue to final destination by same route as secreted proteins (ER - Golgi - plasma membrane or lysosomes), but are transported along this route as components of membrane instead of soluble proteins.
- Integral membrane proteins embedded in membrane by hydrophobic regions that cross lipid membrane
- Regions of protein that. Cross the lipid bilayer are usually α-helix regions made of up to 20-25 hydrophobic aa
- Formation of α-helix:
- maximises H bods between peptide bonds
- hydrophobic side chains of aa interact with fatty acid tails of phospholipids
13
Q
- How various integral membrane proteins differ in how inserted
8. 1
A
- Some cross membrane once, others have multiple regions that cross membrane.
- Some proteins oriented with carboxyl-terminal end on cytosolic side; others have amino-terminal exposed on cytosolic side.
- Most inserted via cotranslational pathway.
- Orientation of inserted protein membranes is established as growing polypeptide chains translocated into ER.
- ER lumen is topologically equivalent to outside of cell, so domains of plasma membranes that are exposed on cell surface correspond to regions of polypeptide chain that are translocated inside ER.
14
Q
- Internal transmembrane sequences
8. 1
A
- Many proteins inserted directly into ER membrane via these sequences. Recognised + translocated by SRP, but not cleaved by signal peptidase. In these cases no amino-terminal signal recognised by SRP, but instead recognises internal (transmembrane) signal sequence
- Helicase chain exits translocon + binds the protein to ER membrane
- Hydrophobic transmembrane sequence indicates change in translocon, causing hydrophobic transmembrane domain of protein to exit translocon into lipid bilayer
- Depending on transmembrane signal sequences, proteins inserted into membrane by this mechanism may have amino or carboxyl end exposed in cytosol
15
Q
- How do transmembrane sequences affect which end is exposed in the cytosol
8. 1
A
- A) Internal transmembrane sequence directs insertion of the polypeptide so amino (N) end is exposed on cytosolic side:
- Transmembrane sequence exits translocon to fix protein in lipid bilayer and rest of chain is translocated to ER as translation proceeds.
- B) Internal transmembrane sequence directs insertion of polypeptide so its carboxyl terminus (C) is exposed on cytosolic side:
- Other internal transmembrane sequences oriented to direct transfer of amino-terminal part of polypeptide across membrane. Continuous translation produces a protein with its N end in lumen + C end in cytosol.
- C) Insertion of membrane protein with cleanable signal sequence + an internal transmembrane sequence:
- Signal sequence is cleaved when chain crosses the membrane. N end is exposed in ER lumen. Translocation interrupted when translocon recognises a transmembrane sequence. Protein exits translocon laterally + binds to ER. Translation continues + C end remains on cytosolic Sid.
