1. structure & function of eukaryotic organelle

2. Eukaryotic cells have membrane-bound subcellular organelles
nucleus (membrane bounded) , endoplasmic reticulum, Golgi complex, lysosome, peroxisome, mitochondria, vesicles & vacuoles…
contain different molecules

3. The Nucleus contains most of the cell's genetic materials
contains two distinct membranes -- outer and inner nuclear membrane.

4. the outer nuclear membrane
continuous with the endoplasmic reticulum (extension of ER)
ribosomes attached
perinuclear space -- the space between the outer and inner nuclear membranes is continuous with the RER lumen.

5. Endoplasmic Reticulum, ER
SER & RER -- can share the same lumen but different in apearance and function.

6. Smooth endoplasmic reticulum, SER
tubules/vesicles
has no attached ribosomes and can not synthesize proteins.

7. Functions of SER calcium storage (sarcoplasmic reticulum, etc.)
detoxify drugs (by hydroxylation )
decompose glycogen→ glucose
synthesize lipids

8. Rough endoplasmic reticulum, RER
RER results from the presence of ribosomes that are bound to the cytosolic side of the ER’s membrane.
flattened cisternae

9. Functions of RER secretory proteins / membrane proteins synthesis
protein glycosylation, folding and quality control
select secretory and intracellular transport

10. The proteins that are synthesized on the RER should be
those will be secreted
those will be become part of the membrane （integral membrane proteins）
those will be sent to lumen of ER, GC or lysosome

11. The proteins are synthesized by ribosomes.
Ribosome have 2 subunits (large/small).
The conponents of subunit are rRNAs and ribosomal proteins.

13. The protein synthesis needs
ribosome
mRNA
tRNA
amino acid
3 nucleotide codon reading
translate mRNA into a polypeptide chain

14. Protein translocation need signals (signal hypothesis)
G. Blobel and D. Sabatini proposed some translating proteins can be guided to RER by special extra amino acid (signal peptide) at N-terminal end.
Those proteins without signal sequence will be completely synthesized in the cytosol .
G. Blobel, the 1999 Nobel Prize winner

15. Signal Hypothesis
protein synthesis of all proteins begins in the cytosol.
The leading end of the nascent polypeptide chain consists of a signal peptide.

16. Signal Hypothesis
When the leading end emerges from the ribosome, it can be recognized and bound by signal recognition particle (SRP).

17. Signal recognition particle, SRP
composed of 6 distinct polypeptides bound to 1 RNA molecule
a protein-RNA complex with GTPase activity.

18. Signal Hypothesis
SRP can recognize and bind the signal sequence, and can also bind SRP receptor (SRPR) on RER membrane.
After SRP bind ribosome, protein synthesis is suspended.
Both SRP(P54) and SRPR have GTPase activity.

19. Signal Hypothesis
When the complex of ribosome and SRP encounters ER membrane, the SRP binds SRPR, working as docking protein.
SRPR transfer polypeptide chain to translocan channel, then GTP is hydrolyzed and SRP is released.

20. Signal Hypothesis
Protein synthesis resumes while the signal peptide is removed by signal peptidase. Now the polypeptide passes into the lumen (Protein glycosylation happens during this process).

21. Signal Hypothesis
After translation is completed, the ribosome dissociates from RER membrane.
signal hypothesis

22. Go to lumen of ER--Soluble proteins

23. Protein membrane insertion
TypeⅠ
Need T-terminal cleavable signal peptide and stop transfer anchor sequence.

24. Different types of stop transfer anchor sequence
TypeⅡand Ⅲ
protein insertion are caused by internal membrane-spanning sequence, without N-terminal signal peptide.

25. Several start/stop transfer anchor sequences
Multi-pass membrane proteins

26. Protein glycosylation in the RER
On the lumen side of RER membrane, carbohydrates are preassembled to form oligosaccharides unit.
Then unit is transfer to asparagine (Asn) residue of polypeptide chain.
The linkage is happened at amide nitrogen of Asn . (N-linked glycosylation)
recognition sequence (necessary but not sufficient): Asn-X-Ser/Thr

27. Protein glycosylation in the RER
The unit contains 14 residues at first, then 3 glucose and 1 mannose residue will be removed in ER.
Glycosylation makes proteins stable and soluble.

28. Vesicle transport
Vesicular trafficking allows lipids/proteins/luminal contents to reach their destination organelle.
Budding → vesicle formed → move to destination →fuse with membrane

29. The vesicle need the coat proteins assembled on the outside surface of membrane to select the correct cargo.
The adaptor proteins provide the linkage between coat proteins and receptors/cargoes.
proteins contain sorting signals

30. Budding
Need the small GTP-binding protein --a GTPase that can hydrolyze GTP.

31. the small GTP-binding protein
acts like a molecular "switch" that flips between an activated (membrane embedded GTP-bound) form and an inactive (soluble GDP-bound) form.

32. the small GTP-binding protein
Inactive form can be attracted to the ER membrane and activated by guanine nucleotide exchange factor(GEF) --a transmembrane protein residing in the ER membrane.

33. Budding
Once GTPase is bound to the membrane the other coat protein components bind the membrane sequentially.
After vesicle release, the GTP hydrolyzed and the coat complex will be dissociated.

34. At least 3 types proteins can work as coat proteins
COPII : transport from ER → Golgi
COPI : retrograde traffic from GC→ER
clathrin : vesicle release from GC → lysosome/ cell membrane

35. ER → Golgi Transitional ER region– no ribosomes binding
Need COPII coat complex

36. ER → Golgi
Inactive GDP-bound protein Sar1 (Arf1 in COPI ) is activated after bind GTP by Sec12 (GEF)
Sar1 changes conformational and then inserts into the ER’s membrane.

37. ER → Golgi
the coat proteins-- Sec23/24 and Sec13/31 are recruited. These proteins simultaneously contact Sar1p and cargo proteins.

38. ER → Golgi
The Sec23/24-Sec13/31-Sar1 complexes then coalesce to form a much larger complex. This network deforms the membrane enough to bud a vesicle off.

39. ER → Golgi
After vesicle released, the GTP hydrolyzed by Sar1 and the coat complex will be dissociated.
Released vesicles aggregate together and move along the MTs from ER to GC.

40. Rab GTPases ensure the specificity of vesicular docking
Vesicle-surface markers that direct vesicles to the correct place.

41. Rab GTPases ensure the specificity of vesicular docking
Rabs cooperate with SNAREs

42. SNAREs mediate vesicle fusion
SNAREs are integral-membrane proteins that pull membranes together.
The coiled-coil is a tightly intertwined set.

43. 2013 Nobel Prize in Physiology or Medicine
James E. Rothman
Randy W. Schekman
Thomas C. Südhof
2013 Nobel Prize in Physiology or Medicine

44. Golgi apparatus /Golgi complex, GC
It is made up of several vesicles and a stack of flattened cisternae.
structure of GC

45. Polarity structure of GC
forming face: the side face to the ER

46. flattened cisternae is main part of GC

47. Polarity structure of GC
mature face: face towards the cell membrane

48. Functions of GC Modification of proteins
Sorting and distributing proteins to next stop.

49. The end