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Lecture 2
Protein Glycosylation
and Vesicular Sorting
Subcellular Fractionation Can be Used to Distinguish
Between PMP and IMPs
I
Lysate
P13 P100 S100
Msb2
Dpm1
Pgk1
J
Buffer
NaCl
Urea
Na CO
SDS/Urea
Density Gradient Centrifugation Can be Used to Distinguish
Between PMP and IMPs
Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008)
Protein modification by
Glycosylation
The term "CDG-Syndrome“ stands for Congenital Disorders of
Glycosylation, and describes an inherited metabolic disorder in
which protein-glycosylation – the synthesis of carbohydrate-protein
complexes – is impaired.
Protein-glycosylation is a vital process, which plays an important
role in a large number of biochemical and cellular processes.
Cystic Fibrosis
Emphysema
Proteins are
Glycosylated in ER and
Golgi
-important for folding
-important for function
-important for targeting
N-linked on Asn and O-linked on Ser/Thr
Biosynthesis of the Glycosyl Moiety
Tunicamycin
Streptomyces compound
that blocks transfer of
GlcNAc-1-P from UDPGlcNAc to dolichyl-P
Two ways to show that a protein is glycosylated
Tunicamycin - ADDED TO CELLS
Endo H - a glycosidase that cleaves high mannose and
some hybrid N-linked oligosaccharosides
ADDED TO PURIFIED PROTEIN
Sialic Acid glycosylation of Different
Proteins
Proteins can be Modified by
GPI anchors
BiP or calnexin is a chaperone
That prevents protein exit
Until folding is complete
(possibly by covering up exit signals)
CFTR Cl- ion channel receptor in cystic fibrosis patients
Has a mutation that prevents it folding. It is trapped in the ER.
Were it allowed to exit, it would function properly.
Protein Folding by Chaperones
Model of calnexin action. As a nascent polypeptide chain enters the ER, certain Asn residues are glycosylated through the addition of an
oligosaccharide of composition Glc3Man9GlcNAc2. The outermost two glucoses are rapidly removed through the action of glucosidases I and II
to reveal the monoglucosylated species recognized by the lectin sites of calnexin/calreticulin. In their ATP-bound state, calnexin bind to the
monoglucosylated oligosaccharide and hydrophobic segments of the unfolded glycoprotein (via their polypeptide binding or chaperone sites).
Glycoprotein dissociation involves not only the action of glucosidase II to remove the terminal glucose residue but also a change in affinity of the
polypeptide binding site, possibly regulated by a shift from an ATP-bound to an ADP-bound or unbound state. After dissociation, if folding does
not occur rapidly, the glycoprotein is reglucosylated by another ER enzyme, UDP-glucose:glycoprotein glucosyltransferase. This enzyme only
reglucosylates non-native protein conformers. The glycoprotein can then re-bind in dual fashion to the ATP form of calnexin/calreticulin. In this
model, both the glucosyltransferase and calnexin/calreticulin act as folding sensors. The function of this binding and release cycle is three-fold: 1)
the polypeptide binding (chaperone) site prevents glycoprotein aggregation, 2) the lectin and polypeptide sites retain non-native conformers in the
ER until a native structure is acquired (quality control), and 3) calnexin and calreticulin bring ERp57 into proximity with the non-native
glycoprotein. ERp57 catalyzes disulfide bond formation and isomerization within the glycoprotein.
Protein Misfolding leads to
Aggregation and Disease
(prions and Altzheimer’s)
Unfolded Protein Response
Taking out the Trash
Unfolded Protein Response
Ire1p is a transmembrane serine-threonine kinase,
oriented with the amino terminus (N) in the ER
lumen and the carboxyl terminus in the cytosol.
When unfolded proteins accumulate in the ER,
Ire1p oligomerizes, trans-autophosphorylates via
the cytosolic kinase domain (K) and activates the
endonuclease in the tail domain (T).
The endonuclease Ire1p cuts HAC1 mRNA at two
sites, removing a nonclassical intron; the two
exons are rejoined by Rlg1p (tRNA ligase).
HAC1u (‘uninduced’) is not translated owing to
the presence of the intron, and Hac1pu is not
produced (brackets). After Ire1-mediated splicing,
HAC1i mRNA is efficiently translated into Hac1pi,
a transcriptional activator that upregulates
expression of UPR target genes after binding to
the unfolded protein response element (UPRE) in
the promoters of genes encoding ER-resident
chaperones and other proteins.
Vesicular Trafficking allows
Proteins and Vesicles to Reach their Destinations
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are needed to see this picture.
The orientation of transmembrane proteins
Proteins IN the ER face the OUTSIDE of the cell
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COPII = ER-> Golgi
COPI = Golgi to Golgi