Outline

• Biosynthetic Pathway (Post ER)
• Retention and retrieval
• ER targeting (KDEL, KKXX)
• Sorting complexes (for the endomembrane compartments)
• Gycosylation in Golgi
• Flow thru Golgi: vesicle & maturation
• Targeting to lysosomes (M6PR)
• Processing of propeptides
• Conceptually, part of the synthesis of a protein is its proper targeting. Targeting and final processing of most secretory proteins take place in the ER. Of course ER proteins are finished and just need to stay where they are. We will discuss targeting in general terms first then use those principles to understand targeting the the ER, then move on to targeting signals for elsewhere in the cell using the same paradigm, then discuss flow and processing in the Golgi.

Retention and Retrieval (see diagram)

• helps to consider PM as default
• 2 modes of sorting:
• retention mechanism poorly described, few examples
• retrieval is much better understood but still in its infancy
• paradigm of coat-receptor-signal applies throughout the cell

Targeting to ER (see diagram)

• many ER proteins contain C-term sequence KDEL
• escape ER in COPII and are retrieved from ERGIC by KDEL receptor/COPI
• another class of receptor is known: KKXX binds COPI: cargo unclear
• how is receptor uncoupled from cargo? pH, calcium?

Sorting Complexes

• using the retrieval paradigm the action of sorting signals can be understood
• there are many gaps in our understanding

Now we are ready to move out of the ER. Processing must be finished in the Golgi:

• 2 types:
• glycosylation: carried out sequentially by subcompartmentalized enzymes
• cleavage to activate some proteins synthesized as pre-pro-peptides (in TGN & SV)
• what is glycosylation good for?
• folding
• protection
• cell/cell interaction
• targeting
• GlcNAc2-Fuc1-man3-GlcNAc3-Gal3-NANA3 is representative product (see diagram)
• start w/ GlcNAc2-man8
• in cis
• manI removes 3 man
• in med
• NAGTI adds GlcNAc
• manII removes 2 man
• NAGTII adds 2 GlcNAc
• Fuc t’ase adds 1 fuc
• in trans
• GT adds 3 Gal
• ST adds 3 NANA

Targeting to lysosomes provides example of use of glycosylation: see diagram

• key step is recognition of lysosomal enzyme targeting signal by:
• GlcNAc phosphotransferase in cis Golgi
• targeting signal is distributed across proteins
• enzyme has to binding sites: one for protein one for UDP GlcNAc
• uridine then removed by phosphodiesterase (cis?)
• next important step is binding by M6PR in TGN
• pH 6.5 favors binding
• receptor contains tyrosine signal in tail: interacts w/ clathrin coat
• complex is then transported to late endosome
• transport by vesicle that buds & fuses
• pH 5.5 in LE favors dissociation
• phosphate is actually removed to terminate signal
• receptor is then retrieved to the TGN
• enzyme may then be activated by cleavage & transported to lysosome

Flow through the Golgi is probably, at least in part, by maturation (see diagram)

• means new cisternea forms at cis, moves through, and falls apart at trans
• illustrates importance of retrieval, probably COPI
• how TMDs mediate retrieval is not known

Five paths out of TGN (at least)

• constitutive (see diagram)
• default!
• calcium-indep. fusion
• may involve processing of proteins eg. proalbumin by furin
• cleaves at dibasic residues
• regulated (see diagram)
• aggregation for sorting (lumenal pH and calcium)
• receptor unknown
• cytoplasmic calcium-dep fusion w/ PM
• typically involves processing eg. insulin by PC3, PC2, and CP
• basolateral
• tyrosine receptor/signal,
• no lumenal signal known
• apical
• carbohydrate
• GPI anchor
• late endosome

Key points

• Golgi stucture/function
• subcompartmentalization
• Glycosylation sequence
• Maturation vs vesicle transport model
• Sorting from TGN

Examples

• Golgi inheritance?

Golgi structure

• stacks/cell= few to >1000
• cisternae/stack= 2 to >8
• cisternae
• 0.5-1µm, dilated rim and flattened core
• subcompartments
• CGN, cis, medial, trans, TGN
• CGN and TGN are sorting stations

Golgi processing

• proteolytic
• amino acid modification (hydroxylation of K & P in collagen)
• N-glycoslylation finishing steps
• O-glycosylation
• synthesis of complex polysaccharides including GAGs
• phosphorylation, eg mannose phosp for lysosome targeting

Glycosylation

• mannI (cis) removal of 4 manns (man 9 to man 5)
• NAGTI (med) addition of NAG (to man 4 NAG 1)
• mann II (med) of 2 manns (man 5 NAG 1 to man 3 NAG 1)
• NAGTII (med) addition of NAG (to man 3 NAG 2)
• fucosyl and galactosyl transferases (trans) add fucose and galactose
• sialyl transferase (trans) adds sialic acid

Transport within the stack

• maturation model
• algal scales
• vesicle transport model
• Rothman assay

Sorting from TGN

• plasma membrane (BL and apical)
• late endosome
• regulated granule