Intracellular Protein Sorting and Transport

remember:

• polypeptides synthesized at ribosomes
• ribosomes located in the cytoplasm (whole cell except nucleus, plasma membrane, cell wall or extracellular matrix)
• but ribosomes are more specifically located in the cytosol (cytoplasm except all membrane surrounded compartments)

But proteins occur in all compartments!

How do they get there?
And how does the cell know in which compartment they belong?
(Remember: protein composition very different in different compartments)

 
proteins are either synthesized on free ribosomes in the cytosol
or they are synthesized on the membrane-bound ribosomes of the rough ER (Fig. 9.3)

 the membrane-bound ribosomes of the rough ER synthesize proteins that are localized in:

• ER
• Golgi
• Lysosomes
• Plasma membrane
• cell exterior

 

• Cytosol
• Nucleus
• Peroxisomes
• Mitochondria
• Chloroplasts

 

 How is the cell able to differ which protein should be translated from membrane-bound ribosomes and which from free ribosomes?
 

Proteins are targeted to the ER by a signal sequence in their amino terminus!

 Experimental evidence for signal sequence in secreted proteins:
-- in vitro translation of mRNAs on free ribosomes yielded slightly larger proteins.
-- If rough ER was added to test tube: cleavage to normal size and location in ER
 
 

Mechanism of targeting proteins to ER: (Fig. 9.7)

 Signal sequence (~20 AA) usually at amino terminus of polypeptide
------ first part to emerge from ribosome

• 1. while emerging, signal sequence is specifically recognized and bound by signal recognition particle (SRP) which consists, next to polypeptides, of a small cytosolic or scRNA. Binding inhibits further translation

 

• 2. complex binds to SRP receptor on ER membrane

----- targeting

 

• 3.
• receptor releases free SRP
• ribosome binds to protein translocation complex localized in ER membrane
• signal sequence is inserted into membrane channel of protein translocation complex
• 4. translation is resumed with polypeptide growing through channel,

during translocation (growth through channel) signal sequence is cleaved by signal peptidase

 

• 5. complete polypeptide released into ER lumen

 

Note on the side:
major proteins of translocation complex closely related to proteins that translocate polypeptides through plasma membrane in E. coli
------ old and highly conserved secretion process
 
 

Proteins localized in membranes of ER, Golgi, lysosome and plasma membrane

• same transport route like water-soluble proteins, but travel as membrane components (Fig. 9.9, note the orientation that is preserved during transport: protein part exposed to lumen will end up exposed to cell exterior)
• inserted into ER membrane while synthesized

protein folding and processing in ER

translocation into lumen as unfolded polypeptide chain

 folding: (like for proteins synthesized in cytosol) assisted by molecular chaperones
especially from the HSP70 family (HSP70 means heat shock protein of about 70 kD, protein family is always present but shows higher expression during stress that leads to increased protein denaturation like elevated temperatures)

 formation of disulfide bonds: facilitated by protein disulfide isomerase (disulfide bonds possible because oxidizing athmosphere in ER, different from cytosol where athmoshere is reducing and therefore reduced group -SH instead of oxidized group -S-S-)

 protein glycosylation: means addition of sugar residues. Specifically, a complex oligosaccharide is added to an asparagine side chain (N-linked glycosylation, because at -NH2 group of asparagine) in consensus sequence Asn-X-Ser/Thr (X stands for any amino acid residue).

From ER to Golgi:

transport in vesicles via bulk flow (no signal necessary)
instead signal sequence necessary for retention in ER (most commonly Lys-Asp-Glu-Leu or KDEL at carboxyl terminus, KDEL stands for the 4 amino acids in their single letter code)
membrane-bound proteins are directly retained, soluble proteins are retrieved from Golgi
 
 

In Golgi:

• arrival of vesicles at cis Golgi network, sorting for recycling to ER or further transport to Golgi stack
• in Golgi stack: further protein processing
• via addition of simple oligosaccharides to serines or threonines that lie in a specific sequence context (O-linked glycosylation, because addition at -OH group of serine or threonine).
• Some N-linked oligosaccharides are modified to carry mannose-6-phosphate at their ends ---- targets proteins to lysosome. Specific signal in protein provides information for necessary mannose-6-phosphate modification.
• Secreted and plasma membrane proteins extensively modified at their N-linked oligosaccharides when traveling from cisterna to cisterna (but depends on modifying enzymes present in Golgi stack and accessibility of oligosaccharide rather than on a specific signal sequence in the protein)
 
• in trans Golgi network: final sorting!
• If no specific signal: in vesicles with bulk flow to plasma membrane.
• Specific sequence necessary for retention in Golgi.
• Specific sugar signal (mannose-6-phosphate) for lysosome targeting.
 

If proteins synthesized on free ribosomes
folded and processed in cytosol
transported to

• nucleus: specific amino acid sequence called nuclear localization signal that direct their transport through the nuclear pore complex (signal is internal sequence, is not cleaved)
• mitochondrion and chloroplast: signal sequence at amino terminal end (cleaved following import)

But protein transport into mitochondria and chloroplasts actually more complex, because several localizations possible (remember that these organelles are surrounded by two membranes: contain more than one compartment!)