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

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

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)

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

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

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. 1994 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. 1996 Jul;109 ( Pt 7):1975-89

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. 2003 Oct 10;278(41):40262-71

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. 2003 Aug;14(8):3482-93

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

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

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

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)

The Dynamic Nature of the Golgi Apparatus

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

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)

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)

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)

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)

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

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)

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

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)

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

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)

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

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

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)

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

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. 2010 Nov;17(11) :1292-7.

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

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

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

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

Widefield vs. confocal microscopy

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

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

The Basic Principle of Confocal Microscopy

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