structure & function of eukaryotic organelle

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

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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.

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.

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).

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

Go to lumen of ER--Soluble proteins

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

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

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

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

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.

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

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

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

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.

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.

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.

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

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

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.

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

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.

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.

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

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

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

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

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

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

flattened cisternae is main part of GC

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

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

The end