Lecture 11 Flashcards
what is glycosylation?
- where are proteins more glycosylated?
- what are the two types of glycosylation in the ER
- glycosylation of protein = adding sugar chains to proteins
- most soluble and
transmembrane proteins in the ER are glycosylated (in the cytosol it’s rare to be glycosylated)
two types of glycosylation in ER:
- O-linked glycosylation (10%) – adding the sugar group to an oxygen in the amino acid side chain of the protein
- N-linked glycosylation (90%) (very specific) – adding sugar molecule very specifically to the nitrogen on the asparagine amino acid
- N-linked oligosaccharide precursor is performed in the ER
- precursor composes of an N-acetylglucosamine, mannose, and glucose sequence
➢this precursor binds to the Nitrogen on the asparagine side chain on the polypeptide
how does N-linked Glycosylation occur in the ER
- what enzyme facilitates the glycosylation
- what are the two acceptable amino acid sequences the enzyme adds the precursor to?
- In the ER lumen, an N-linked
oligosaccharide precursor is
transferred by an oligosaccharyl transferase to an Asn-asparagine on a protein being synthesized on the ER side - the enzyme detects an Asn-X-Ser or Asn-X-Thr where X is any amino acid except proline, and adds the sugar chain
- proteins are only glycosylated on the ER lumen side
how are N-linked oligosaccharides processed in the ER
REVIEW DIAGRAM
After transfer of the N-linked
oligosaccharide to the protein:
1. three glucoses removed (1 by glucosidase I and 2 by glucosidase II - but one at a time)
➢ this is done for proper folding of the protein
2. 1 mannose removed (by ER mannosidase)
- this glycosylated protein is
transported via vesicles to the
Golgi
how are N-linked Oligosaccharides processed further in the golgi
- there are Cis, Medial, and Trans cisternae in Golgi
- each cisternae has
different [enzymes] - each remove or add different sugars
- result in different
modifications to different
proteins – creates unique processed glycosylated protein
why does glycosylation occur? (4)
- Tag to mark the state of protein folding
- Protect proteins on the cell surface from proteases
- Some glycosylated proteins have a role in cell adhesion
- Allows proteins to form the correct 3D-structure
how is glycosylation used to tag to mark the state of protein folding
- essentially the process of glycosylation can ensure that a protein is correctly folded in the ER before it exists to the golgi
how?
- precursor oligosaccharide is added to the unfolded protein at the correct AA sequence as it exists the ribosome
- glucosidase I removes one glucose
- glucosidase 2 removes one glucose
- before glucosidase 2 removes the third glucose the following occurs:
–> calnexin, a TM chaperone protein, binds to the precursor oligosaccharide’s remaining glucose and helps the unfolded protein to fold.
–> once folded, glucosidase 2 removes the third glucose and ER mannosidase removes a mannose sugar
- from here, if the protein is properly folded, it exits the ER
- however, if not properly folded, glycosyl transferase enzyme adds a glucose via UDp –> UDP - glucose transport
- the addition of the glucose again gives calnexin chaperone another shot to properly fold the protein again
Third method of protein sorting: Vesicular transport
how do vesicles move cargo proteins between compartments (3 simple steps)
- vesicle is formed in the ERs donar compartment
- budding occurs
- vesicle fuses with target compartment
Cargo proteins are delivered by transport vesicles in vesicular transport.
what are the three cargo proteins involved in vesicle transport?
- 3 Cargo proteins:
➢ transmembrane proteins (TM is green in diagram - stays consistent across the membrane)
➢ soluble proteins (red in diagram, free in lumen) - Note: some soluble cargo proteins are bound by transmembrane cargo receptors (blue that i drew - picks up soluble proteins via one end, stays consistent, releases cargo when fused)
Topic 1. How do vesicles form
- which vesicles have protein coats
- what do protein coats do (3)?
- to make vesicles you need protein coats
- new transport vesicles have protein coats – old vesicles do not have protein coats
what do they do?
- they select cargo for vesicle
- they give curvature to vesicle
- they promote vesicle budding
- then after the vesicle is made and the top three functions are done, they get rid of the protein coat
what are three protein coats involved in vesicle budding?
- where do they form and transport to?
COPI-Coated Vesicles
* from Golgi to ER
* between different Golgi cisternae
COPII-Coated Vesicles
* from ER to Golgi
Clathrin-Coated Vesicles
* from Golgi apparatus and
plasma membrane to endosome
see diagram slide 16
how are monomeric GTPases used to make vesicles
- GTPases are involved in creating vesicles
monomeric GTPases cycles between:
* GDP-bound (OFF)
* GTP-bound (ON)
if you get signal: need to make vesicles
- GEF (guanine nucleotide exchange factor) is used to transform GDP to GTP on GTPase
- this will stimulate formation of vesicles
if you get signal: stop making vesicles
- GAP (GTPase-activating protein) is used to transform GTP to GDP on GTPase
- this will stimulate inhibition of vesicle formation
Steps in coat assembly to form vesicles:
1. Send an initial signal using GTPase to recruit coat proteins
which coat proteins use which type of GTPase?
how is the first step done?
Different coat proteins have different types of GTPase for recruitment:
* COPI, clathrin-coated vesicles
➢ enzyme: ARF GTPase
* COPII-coated vesicle
➢ enzyme: Sar1 GTPase
ex. formation of COPII-coated vesicles (same steps for any coat)
note: Sar1-GDP is the GDP-bound form of the GTPase Sar1.
1st step:
- there is an inactive Sar1-GDP with an amphiphilic helix attached
- the Sar1GDP binds to the Sar1 GEF which is bound on the ER membrane
- the GEF switches GDP to GTP and conducts a conf change by exposing the amphiphilic helix so it can bind to the membrane and become active.
- this recruits coat protein subunits
Steps in coat assembly to form vesicles:
2. recruit coat proteins and create vesicle bud
what are the two vesicle coat layers?
what are the three things the coat proteins need to select?
- next step is to select cargo and start forming the bud
- Inner layer
➢ binds to membrane and selects cargo - Outer layer
➢associates with the inner layer to promote
polymerization of the coat (sometimes also selects
cargo)
Coat proteins need to select:
* Cargo (transmembrane proteins)
* Transmembrane cargo receptors (y-shaped receptors which bind soluble free cargo proteins)
* SNAREs
COPI-Coated vesicles
- what are the inner and outer proteins it selects
- what do these proteins do
- what are the three steps to uncoating
- Inner: 4 subunits (β γ δ ζ)
- Outer: 3 subunits (α β’ ε)
- select specific cargo
- vesicle is curved and fully formed
Uncoating:
1. γ-COP (inner) binds to Arf GAP is one of the proteins on the membrane
2. GAP causes GTP hydrolysis (Arf-GTP → Arf-GDP)
3. Arf-GDP detaches from membrane and thus all the coats release
COPII-Coated vesicles
- what are the inner and outer proteins
- what do these proteins do
- what are the three steps to uncoating
same as COPI but change names
- Inner: 2 subunits (Sec23/Sec24)
- Outer: 2 subunits (Sec13/Sec31)
- select specific cargo
Uncoating:
1. Sec23 (inner) has GAP activity
➢ stimulated by Sec13/Sec31 (outer)
2. GTP hydrolysis (Sar1-GTP → Sar1-GDP)
3. Sar1-GDP detaches from membrane (helix not exposed) and all coats are released
