WEEK 3 - GOLGI APPARATUS, VESICLE TRANSPORT, LYSOSOMES Flashcards
GOLGI APPARATUS, VESICLE TRANSPORT, LYSOSOMES
Golgi apparatus
- made up of citernae and sheets
- generates vesicles (+ lysosomes)
- modification and packaging o proteins and lipid for exocytosis
- made of cisternae
- the Golgi is a stack of flattened membrane bound compartments (cisternae)
- The cis face lies nearest the ER and is the site at which vesicles from the ER dock
*The trans face is the site from which vesicles depart for the cell surface or other compartments
Golgi structure
endoplasmic reticulum
golgi vesicles
vesicular tubular cluster
cis Golgi network (CNG)
cis cisterna
medial cisterna
trans cisterna
trans Golgi network (TNG)
secretory vesicles
plasma membrane or other organelles
functions of the Golgi
- proteins destined for secretion and for a variety of organelles/vesicles within the cell are sorted, modified and dispatched from the Golgi apparatus
- it is also a major site of carbohydrate synthesis in the form of glycoprotein and proteoglycans
- therefore the Golgi tends to be prominent in secretory cells like this goblet cell
Dispatch from the endoplasmic reticulum to the Golgi
- Almost all proteins in the ER lumen are glycosylated and this acts as a folding tag
- During the initial phases of folding 2 of the 3 terminal glucosemolecules are removed
- Calnexin is a chaperone which recognises the single glucose of incompletely folded proteins and prevents their export to Golgi
The final glucose is removed then :
1. If partially folded, glucosyl transferase adds another glucose and it tries again
2. Misfolded proteins are chaperoned back to the ER protein translocator and are sent to the cytoplasm for degradation
3. Correctly folded proteins go on to be exported to the Golg
Fully folded proteins are targeted to ER exit sites
- fully folded proteins are stored in a particular part of the ER that forms a bud
- For soluble proteins this involves interactions with transmembrane receptors
- Proteins need “exit” signals for efficient export: non-cargos are packaged at a much lower rate
- Most exit signals are not known but:
- ERGIC53 receptor protein binds to mannose on Factors V and VIII
- ERGIC53 deletion → haemophilia (factor V and VIII involved in blood clotting)
- The coat protein COPII interacts with the cytosolic tail of the receptor causing a vesicle to bud off (details next lecture)
Movement of the transport vesicles
- Once COPII coated vesicles bud from the ER they rapidly shed their coat
- They undergo homotypic fusion –like joining with like (Details of how vesicles fuse next set of lecture bites –vesicular transport)
- The resulting vesicular tubular cluster (VTC) moves along microtubules (see cytoskeleton lectures –week 4) to mature (deliver) its contents to the Golgi
- VTC can regulate its pH as it has proteins in the membrane that allow H ions to enter, increase in H ions causes the pH to go down (acidic)
- acidic environment causes the receptor to change shape since some aa change their charge bc they in a different pH, receptor and cargo separate once they reach the Golgi
- Once the cargo reaches the Golgi it is released from its receptor, Mediated by a decrease in pH
Why might the change in pH cause the cargo to be released from its receptor?
- pH change can change the charge of aa depending on its pKa which will dissociate the receptor holding on
Transport back to the Endoplasmic Reticulum
- ER proteins are retrieved from the Golgi
- Proteins that participate in ER budding (receptors) retrieved from the vesicular tubule cluster (recycled)
- Also proteins which have escaped the ER by mistake need retrieving
- COPI coated vesicles bud from VTC / Golgi, are uncoated & transported back to ER
- Resident ER membrane proteins contain the cytosolic sequence -KKXX which interacts directly with COPI
Modifications: Oligosaccharide processing
- Endoplasmic Reticulum
- N-linked glycosylation
- Folding and glucose trimming
- Cis cisterna
- Mannose trimmed
- Medial cisterna
- Mannose trimmed
- N-acetyl glucosamine added
- Trans cisterna
- Addition of galactose and N-acetylneuraminic acid
Other modifications within the golgi
- O-linked glycosylation (in the cisternae)
- the addition of sugars to the -OH group of Ser, Thror hydroxylysine
- catalysed by glycosyl transferases
- usually N-acetyl galactosamine is added first
- additional residues ranging from a few to over 10 are then added
- Phosphorylation of oligosaccharides on lysosomal proteins (in the CGN)
- further details in lysosomes lecture bites
- Sulphation of tyrosine and carbohydrates (in the TGN)
- increases their negative charge
- these things are here to help order things where the component goes and to make sure its associated with the receptor only under certain circumstances
What is the point of glycosylation?
- As a protein marker
- for complete folding
- for targeting transport between the ER and Golgi
- for sorting within the Golgi
- As a protector
- relatively inflexible oligosaccharides prevent proteases approaching and digesting extracellular proteins
- As a cellular marker
- Oligosaccharides on cell surface proteins form part of the cell-cell recognition and adhesion mechanism. Different cell types may express a slightly different complement of glycosyltransferases, allowing them to modify their surface proteins in a cell specific way
- Regulatory Roles
- Cell surface signalling receptors may be glycosylated and alterations to their glycosylation pattern may affect their activity in different cell type
Transport through and out of the golg
- Vesicles from the ER fuse to form VTCs and Cis Golgi Network
- Once completed CGN displaces upwards and becomes cis cisterna
- matures into the medial
- and then trans cisternae
- Enzymes etc retrieved by retrograde transport
- Vesicles bud from TGN for dispatch or retrieval, until it is all gone and another cisterna replaces it
summary
ER
SORTING
-phosphorylation of oligosaccharides on lysosmal proteins
- removal of Mannose
-removal of Mannose, addition of GLcNAc
-addition of Gal, and NANA
-sulfation of tyrosines and carbhodrates
SORTING
Name the chaperone which monitors protein folding prior to export from the ER to the Golgi
calnexin
Calnexin retains proteins with a single terminal glucose on their N-linked oligosachharide chain and retains them in the ER. Following removal of the terminal glucose: If folding is complete the protein will continue its journey to the Golgi. If folding is not yet complete a new terminal glucose can be added (by glucosyl transferase) and the protein can have another attempt at folding. However if the protein is mis-folded, it must be sent to the cytoplasm through the protein translocator, where it is degraded.
What coat protein causes vesicles destined for the Golgi to bud from the ER?
COPII
What fate awaits a protein containing the KDEL sequence when it reaches the Golgi?
Retrieval to the endoplasmic reticulum
Vesicles which return proteins to the endoplasmic reticulum from the Golgi and coated in
COPI
Name two modifications which can occur to proteins within the Golgi
Glycosylation, oligosaccharide processing, O-linked glycosylation, Phosphorylation, Sulphation
Match the type of glycosylation to the amino acid
- O-linked
- hydoxylysine
- serine
- threonine - Not glycosylated
- alanine
-glycine - N-linked
-asparagine
Proteins which pass through the endoplasmic reticulum and Golgi are glycosylated in many different ways. What are the roles of this glycosylation?
A marker for folding
A transport marker
A cell surface marker
Protection
The Principles of Vesicular Transport
- Membrane bound vesicles carry cargo in their lumen and membrane from a donor to a recipient compartment.
- Vesicles bud from specialised coated regions of the donor compartment.
- The coat is later discarded, allowing the membranes of the vesicle and recipient to interact and fuse
Membrane Coat Proteins
- COPII–coats the vesicles which transport molecules from the ER to the Golgi
- COPI–coats the vesicles which transport molecules from the Golgi back to the ER
- Clathrin–coats the vesicles which transport molecules between the Golgi, the lysosomes and the plasma membrane
Clathrin
- Each clathrin subunit contains 3 large and 3 small polypeptide chains which together form a three legged triskelion
- Triskelions form a basket-like convex framework of pentagons and hexagons on the cytosolic side of membranes
Role of clathrinin transport
- A second protein complex, adaptin, is needed to attach the clathrin coat to the membrane
- Adaptins also interacts with transmembranous proteins, including the transmembranous receptors which capture soluble cargo for transport
- There are at least 4 different adaptins, each recognising a different set of cargo receptors
