Download MTC25 - Intracellular Processing

MTC25: INTRACELLULAR PROCESSING AND TARGETTING
05/10/07
LEARNING OUTCOMES
Describe the role of the ER, Golgi and cytoskeleton in facilitating intracellular transport
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Newly formed polypeptides / proteins are released from ribosomal sites in the nucleus to be
transported to other areas of the cell
Translocation to the rough endoplasmic reticulum (ER) occurs during protein synthesis:
o A signal sequence is formed on the nascent protein which is identified and bound to by a
signal recognition particle (SRP); synthesis is then temporarily halted (translation arrest)
o SRP receptors (docking proteins) on the transmembrane of the rough ER interact with the
SRP complex to ‘hold’ it at an adjacent protein channel (translocon) – only at this point does
synthesis resume (this ensures that proteins are properly translocated)
o The SRP is released from the complex and the nascent protein passes through the translocon
as it develops and into the lumen of the rough ER
o As translation continues, an enzyme called signal peptidase which resides in the ER
membrane cleaves the signal sequence from the polypeptide
o In the case of secretory proteins (not membrane proteins) the polypeptide translocates
completely through the membrane and into the lumen, at which point the ribosome
dissociates
Once inside the rough ER, new proteins undergo a series of post-translational modifications depending
on their ultimate destination and function, including glycosylation of 14 N-linked sugars and formation
of tertiary structure under the supervision of chaperones (a large class of ATP-hydrolysing proteins)
The smooth ER is so called because it is not studded with ribosomal sites like the rough ER and so has
no SRP-related components; it participates in lipid synthesis and detoxification, e.g. of alcohol
The most common genetic defect that causes CF, ΔF508 (deletion of three nucleotides resulting in the
loss of phenylalanine (F) at the 508th position), prevents the CFTR protein from folding properly which
results in its ejection from the ER and subsequent decomposition
Defective proteins are broken down through the ubiquitin-proteasome pathway as follows:
o Ubiquitin (Ub) is activated by a combination of enzymes: activating enzyme E1, conjugating
enzyme E2 and ligating enzyme E3
o The target protein is attacked by the ubiquitin and subsequently degraded by a 26S
proteasome site
o The protein is decomposed into small peptides and the ubiquitin is recycled
The Golgi complex is a stack of disc-like membranes with a cis or forming face which faces the nucleus
and a trans or exit face which is used to sort newly formed proteins for export to other areas of the
cell such as lysosomes, cell surface or secretory vesicles
On entering the cis stack of the Golgi, proteins undergo a ‘remodelling’ of the 14 N-linked sugars
Sugar transferases and mannosidases trim and modify the oligosaccharide chain in a process called
complex glycosylation
By default, when proteins reach the trans face of the Golgi they are packaged in vesicles and taken to
the cell surface; this in an unregulated process called constitutive secretion
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More specialised secretory proteins that must be regulated are packaged in specialised secretory
vesicles which mediate calcium-dependent exocytosis:
o Vesicles are typically coated by clathrin proteins which exist as three-armed ‘tri-skeletons’
that attach to one another around the subject protein, producing a ball-like cage structure
o Electrostatic repulsion makes direct fusion of vesicles to other membranes very difficult, so
the process is mediated by proteins known as SNAPs and SNAREs which bind together very
tightly, allowing fusion to take place
o After fusion, the contents of the vesicle are released, its membrane recycled, and an enzyme
called the NEM-sensitive factor (NSF) separates the SNAP and SNARE for reuse
Lysosomal proteins are marked as such by mannose-6-phosphate (M6P) within the Golgi and then
exported by receptor mediated endocytosis:
o The M6P binds to a receptor in the trans section of the Golgi where a vesicle is formed
around it
o The clathrin coat is shed and the vesicle fuses with an endosome which causes the M6P to be
removed leaving the mature protein package to be transported to a lysosome
o The receptors used organise in a vesicle and are recycled back into the trans-Golgi
An alternative sorting pathway from the ER is through the cytosol, from which proteins can be
translocated to mitochondria, the nucleus, or peroxisomes
For mitochondrial transport, a signal peptide consisting of roughly 24 amino acids flags the subject
protein which is then ‘threaded’ across both mitochondrial membranes simultaneously (and its
tertiary structure restored within), after which the signal peptide is cleaved leaving the mature protein
The nucleus has a double membrane with nuclear pore complexes that house nuclear pores roughly
9nm in diameter