1. Localization and Retention of Glycosyltransferases And the Role of
Vesicle Trafficking in Glycosylation
Richard Steet, Ph.D.
2/26/2013

2. compartmentalization and localization of glycosylation enzymes
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
enzyme specificity
availability of substrates
compartmentalization and localization of glycosylation enzymes

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

4. Important caveats regarding the intrinsic mechanisms…
Observations made with one enzyme are not necessarily applicable to others
Golgi-retention properties of any given glycosyltransferase may vary depending
on the cell type
Variations in the expression level of a glycosyltransferase in an experimental
system can have MAJOR influence on its localization/retention
Many studies have used chimeric proteins composed of segments of GTs fused
to reporter protein but conclusions not always verified using intact GTs
In vitro studies using intact Golgi compartments indicate some spatial and
functional overlap among enzymes that were previously thought to be segregated
Almost nothing is known about the mechanisms that govern the localization of
nucleotide-sugar transporters

5. 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)
* search for similar retention sequences in Golgi glycosylation enzymes
has not been conclusive

6. 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 suggesting that enzymes interact
EMBO J Feb 1;13(3):562-74
these enzymes were later shown to associate via their stem regions (helps ensure that these
enzymes act sequentially in N-glycan processing); referred to as “kin recognition”
J Cell Sci Jul;109 ( Pt 7):

7. 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
J Biol Chem Oct 10;278(41):

8. 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
Mol Biol Cell Aug;14(8):

9. 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

10. 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

11. What does the Golgi apparatus actually 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

12. 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)

13. The Dynamic Nature of the Golgi Apparatus

14. TGN: trans-Golgi network CGN: cis-Golgi network
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
TGN: trans-Golgi network CGN: cis-Golgi network

15. cargo: secreted glycoproteins enzymes: nucleotide-sugar
The vesicular transport model views the Golgi as a stable organelle with secreted proteins
being moved between cisternae or stacks in small vesicles
cargo
enzymes
cargo: secreted
glycoproteins
enzymes: nucleotide-sugar
transporters and glycosyl-
transferases
glycosylation enzymes are stable “residents” of the Golgi (this model depends heavily
on intrinsic mechanisms for Golgi retention)

16. modes of retrograde transport: - intra-Golgi (enzyme localization)
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
cargo
enzymes
modes of retrograde transport:
- intra-Golgi (enzyme localization)
- Golgi-to-ER (lipid balancing)
cargo
enzymes
cargo
enzymes
Golgi membranes and glycosylation enzymes are highly mobile (this model depends
heavily on retrograde transport and protein/lipid recycling)

17. The Secretory Apparatus Contains Several Anterograde (forward) and
Retrograde (backward) Pathways
* vesicles that mediate transport in these pathways are often defined
by their coat proteins (i.e. COPI, COPII, clathrin)

18. What is involved in retrograde transport and vesicle trafficking within the Golgi?
recruitment of proteins into COPI-coated vesicles
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)

19. Budding of Vesicles from Donor Membranes and Fusion with Acceptor
Membranes Is a Highly Orchestrated Process

20. What is involved in retrograde transport and vesicle trafficking within the Golgi?
recruitment of proteins into COPI-coated vesicles
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)

21. Recruitment of Recycling GTs into COPI-coated Vesicles
proteins involved in COPI-mediated transport are peripheral Golgi proteins
that cycle between the cytosol and the Golgi membrane - they are not able to
bind to the lumenal portion of GTs
only the short cytoplasmic tails of GTs (10-30 amino acids) are accessible
to these peripheral Golgi proteins
Are there short sequences within glycosylation enzymes (i.e. in their
cytoplasmic portions) that mediate Golgi localization and retention?
studies in yeast have shown that a sorting protein Vps74p can recognize
specific sequences in the cytoplasmic tails of some glycosyltransferases; Vps74p
also binds to COPI subunits

22. What is involved in retrograde transport and vesicle trafficking within the Golgi?
recruitment of proteins into COPI-coated vesicles
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)

23. Vesicle Fusion is Governed by SNARE proteins
SNARE: Soluble NSF Attachment protein REceptor
NSF: N-ethylmaleimide-Sensitive Factor
SNAP: Soluble NSF Attachment Protein

24. What is involved in retrograde transport and vesicle trafficking within the Golgi?
recruitment of proteins into COPI-coated vesicles
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)

25. The movement of proteins between different compartments
is mediated in part by multisubunit protein complexes

26. Structural studies on Dsl1 reveals
its role in tethering and other
aspects of vesicle targeting such
as uncoating and fusion

27. The COG complex acts as an intra-Golgi COPI-coated vesicle tether
Coiled-coil golgin tethers: GM130, p115, giantin
Multisubunit complex tethers: COG, TRAPP, GARP
COPI coated
vesicles
Golgi stack
COG
Rab GTPase
v-SNARE
t-SNARE
COPI coat
Shed COPI coat
adapted from Oka et. al., TICB (2006)

28. Molecular Organization of the COG Complex
the eight subunits of COG are thought to be organized into two lobes

29. Single particle EM studies of the COG Complex reveal a
Y-shaped structure with three flexible, highly extended legs
Molecular organization of the COG vesicle tethering complex.
Lees JA, Yip CK, Walz T, Hughson FM.
Nat Struct Mol Biol Nov;17(11) :

30. loss of COG complex subunits in CHO cells (and in human
patients) cause glycosylation defects
- four LDL-receptor deficient CHO mutants characterized in
mid 80s by Monty Krieger (ldlA, ldlB, ldlC, ldlD)
ldlB, ldlC - pleiotropic effects on glycosylation
abnormal glycosylation of receptors leads to rapid degradation at cell surface
human patients with defects in nearly all the COG complex subunits
have now been identified; the mechanisms whereby loss of COG
complex function affect glycosylation are still being debated

31. How does loss of a functional COG complex affect glycosylation?
the COG complex as a mediator of stability of glycosyltransferase stability
Glycobiology 2010
the COG complex as a tethering factor for specific glycosyltransferase-
containing retrograde vesicles
Traffic 2006

32. 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
details regarding the protein machinery that executes retrograde transport of
glycosylation enzymes within the Golgi and other compartments is slowly emerging

33. two basic types of fluorescence microscopy:
Much of our progress in understanding the Golgi and vesicle
trafficking has been made by visualizing intracellular structures
using fluorescence microscopy
two basic types of fluorescence microscopy:
1) widefield or epifluorescence microscopy
2) laser scanning confocal microscopy

34. Widefield vs. confocal microscopy

35. Differences Between Widefield/Epifluorescence and Confocal Microscopy
Mode of excitation: cone of light in widefield vs. laser focused on a
single point in confocal, the laser scans in the xy plane within a
stack of optical sections (z-series)
2) Collection of light: selective in confocal (pinhole); secondary
fluorescence in areas is removed from the focal plane; much thicker
samples can be imaged
Image can visualized directly in widefield/epifluorescence but must be
reconstructed by a computer in confocal

36. Basic Features of a Confocal Microscope
Contrast and definition are dramatically improved over widefield techniques
due to the reduction in background fluorescence and improved signal-to-noise

37. The Basic Principle of Confocal Microscopy

39. Use of Confocal Microscopy to Determine Co-localization
Of Two Proteins