1. glycosylation is a non-template derived phenomenon
the presence of certain sugars within a given oligosaccharide chain is not
determined by a pre-defined plan (like transcription and translation) but
depends on multiple factors:
- expression level of glycosylation enzymes
- availability of substrates
- localization of glycosylation enzymes
Glycans are generated by biosynthetic pathways comprised
of an assembly line of glycosyltransferases within the Golgi

2. the proper order and in the proper compartment?
How are the glycosyltransferases and nucleotide-sugar transporters kept in
the proper order and in the proper compartment?
multiple mechanisms involved -
1) information is contained within the structure or sequence of the enzymes (intrinsic)
2) dynamics of the Golgi (global)

3. Intrinsic mechanisms: ER retention sequences
the localization of protein O-fucosyltransferase 1 to the ER is mediated by the presence of a KDEL-like sequence in its C-terminal tail
human OFut1: rpssffgmdrppklrdef
fly OFut1: fwgfpkekdrkhtnvheel
(KDEL: classic sequence for receptor-mediated retention of ER resident proteins)
are there short sequences within glycosylation enzymes (i.e. in their cytoplasmic
portions) that mediate Golgi localization and retention? (answer still not clear)

4. Intrinsic mechanisms: physical and functional associations (part 1)
GlcNAcTI was retained in the ER by grafting the cytoplasmic tail of Iip33 to its own
tail; endogenous ManII was also retained in the ER suggseting that enzymes interact
The two enzymes were later shown to associate via their stem regions (helps ensure
that these enzymes act sequentially in N-glycan processing)

5. Intrinsic mechanisms: physical and functional associations (part 2)
pathways of glycolipid biosynthesis
GalT, SiaT1, SiaT2 form
a trimeric complex
Associations between enzymes involved in glycolipid biosynthesis help to shunt
substrates along certain pathways and generate specifically glycosylated
products

6. Intrinsic mechanisms: physical and functional associations (part 3)
GalCer synthase binds to UDP-galactose transporter and selectively retains a
fraction of the total transporter in the ER

7. Intrinsic mechanisms: physical and functional associations (part 4)
GlcA
IdoA
Association of EXT1 and EXT2 produces a highly active polymerase for HS biosynthesis
and is required for Golgi localization
Association of GlcA epimerase and 2OST ensures that IdoA is sulfated, limiting its reversion

8. Global mechanisms: Golgi structure and dynamics
Two main Golgi functions are: SORTING and GLYCOSYLATION
Golgi
trans Golgi
network (TGN) ER
plasma membrane
trans
medial
cis
ERGIC

9. What does the Golgi apparatus really look like and what is it composed of?
electron micrograph
% of total Golgi proteome
Golgi membranes are composed of several different types of lipid molecules; Golgi proteins can be
integral membrane proteins (“resident”) or peripheral proteins that associate with the cytosolic
face of the Golgi

10. How are proteins distributed within Golgi membranes?
immunogold electron microscopy
UCE and CD-MPR: TGN proteins
Although Golgi proteins and enzymes are often concentrated in certain stacks or cisternae,
they really exist in a “distribution gradient” within the Golgi and can also be found in other
subcellular compartments (i.e. endosomes or ER)

11. = Global/intrinsic mechanisms: lipid bilayer sorting Golgi ER
transmembrane domain (Golgi enzyme)
transmembrane domain (cell surface receptor)
Golgi
trans Golgi
network (TGN) ER
plasma membrane
Higher sphingolipid
and cholesterol
thickening of
the membrane =
Since Golgi enzymes tend to have shorter transmembrane regions compared to cell
surface proteins, it was thought that the thinner bilayer of the Golgi would retain
these enzymes and not allow them to reach the plasma membrane

12. What is the true nature of the Golgi apparatus?

13. Vesicular transport vs
Vesicular transport vs. cisternal maturation: competing models of Golgi dynamics
* these two models differ in how secreted proteins move through the Golgi, how Golgi
enzymes are retained within the organelle and the nature of the Golgi itself

14. The vesicular transport model views the Golgi as a stable organelle with secreted proteins
being moved between cisternae or stacks in small vesicles
glycosylation enzymes are stable“residents” of the Golgi (this model depends heavily on
intrinsic mechanisms for Golgi retention)

15. The cisternal maturation model views the Golgi as a highly dynamic organelle that
continuously arises from the ER and is consumed at the trans-Golgi network
modes of retrograde
transport:
- intra-Golgi (enzyme
localization)
- Golgi-to-ER (lipid
balancing)
Golgi membranes and glycosylation enzymes are highly mobile (this model depends
heavily on retrograde transport and protein/lipid recycling)

16. What is involved in retrograde transport and vesicle trafficking?
recruitment of proteins into COPI-coated vesicles (many proteins)
budding of vesicles from membranes (ARFs, Rabs, etc.)
transport of vesicles to target membranes (Rab effectors, dynein, kinesin,
microtubules)
tethering of vesicles to target membranes (multiprotein complexes - TRAPP, COG, GARP)
binding and fusion of acceptor and target membranes (SNAREs, SNAP)

17. mechanisms might contribute to Golgi enzyme localization
An integrated (and therefore complex) view of how both intrinsic and extrinsic
mechanisms might contribute to Golgi enzyme localization
GREEN - late Golgi enzymes
PINK - early Golgi enzymes
Early Golgi enzymes move through the stack by cisternal progression, forming large complexes that
readily recycle back to early compartments
Due to lack of signals or selective partitioning, late Golgi enzymes do not efficiently recycle and thereby
concentrated in the TGN

18. Key Points:
the fidelity of glycan biosynthesis depends, in part, on the order of glycosylation
enzymes within the Golgi apparatus
both structural features of the enzymes and the overall dynamics of the Golgi
contribute to enzyme localization
physical and functional interactions between glycosylation enzymes ensures that
modifications are properly made
the two competing models of Golgi organization, the vesicular transport model and
the cisternal maturation model, differ in how secreted proteins move through the
Golgi, how Golgi enzymes are retained within the organelle and the nature of the
Golgi itself
an understanding of how Golgi glycosylation enzymes are localized and identification
proteins that regulate this process is slowly emerging

19. How important are Golgi dynamics and retrograde trafficking in the
maintenance of proper glycosylation?