Lecture 11 - Protein Sorting Flashcards
What are the 3 mechanisms that proteins can move from one compartment to another?
Gated transport Transmembrane transport Vesicular transport
What is gated transport?
Movement between cytosol and nucleus through nuclear pore complex which act as a selective gate that actively transport certain molecules
What is transmembrane transport?
Movement of proteins across a membrane from the cytosol into a topologically distinct space e.g. cytosol Into ER, cytosol into lysosomes) Use a protein translocator, transporter protein usually has to be unfolded to fit through
What is vesicular transport?
Membrane-enclosed transport packages. Move between different compartments (e.g. ER to Golgi)
Why does targeting occur?
Information needed for the process is embedded in the structure of these proteins: signal sequence
How is the information encoded?
Primary structure (amino acid sequence)
How are targeting information be generated?
Modification to a protein in post-translational maturation event - processing
What removes signal sequence?
Specialised signal peptidase
Endoplasmic reticulum
Occupies much of the volume of eukaryotic cell Largest membrane in cell (large surface area) Continuous with nuclear envelope under EM Site of production of ALL transmembrane proteins and Lipids. Almost all secreted proteins are derived from ER lumen
What are examples of secretory and organelle proteins?
Peptide hormones - synthesised in the endocrine gland and brain (insulin, growth hormone, prolactin, ACTH) Digestive enzymes - synthesised in the pancreas Blood proteins - synthesised in the lifer (albumins, globulins, and clotting factors) Immunoglobulins - synthesised in lymphocytes Collagen - synthesised in fibroblast
When do Ribosomes bind to the ER membrane?
Co-translational translocation
What happens when Ribosomes complete synthesis of protein?
Release it prior to post-translational translocation
Approximately how many peptide bonds are synthesised in free Ribosomes in the cytosol?
70 peptide bonds
What directs the Ribosomes to the ER?
A sequence of N-terminus amino acids
What does the signal peptide allow?
Free Ribosomes to become membrane associated and growing polypeptide to cross the ER
What bridges between the ribosome and the ER membrane that enhance ribosome Binding to the ER?
Mg2+ ions
What part of the protein is highly hydrophobic?
N-terminal 40 residues protrude from the 60S ribosomal subunit
What does signal recognition particle (srp) bind to?
Ribosome and signal peptide
What is necessary for SRP Binding?
The unfolded state of the nascent peptide chain
What does SRP do?
Interacts with ribosome, required for protein
What does SRP contain?
6 discrete polypeptides 7S RNA 300 nucleotides long
What is RNA related to?
Highly repetitive Alu DNA
What are the SRP proteins in S-domain?
S19, S54, S68, S72
What are the SRP proteins in Alu-domain?
S9 and S14
Wher does The SRP bind to the signal peptide?
Via SRP54 in the S domain forming a complex with GTP
What does GTP binding to SRP54 increase for the ribosome
SRP affinity
Where is SSR located?
Adjacent to a protein translocation embedded in the ER membrane
What is signal sequence receptor?
Integral endoplasmic reticulum glycoprotein
What is SSR composed of?
Two subunits SRa and SRb
Where does both subunit (SRa and SRb) bind?
GTP
What does the SRb-GTP complex interact with?
Ribosome-SRP-nascent chain complex
What does the SRb-GTP complex induce?
The transfer of signal peptide to the Translocon
Where does the ribosome fit tightly?
The cytoplasmic side of the pore
What happens in contrast with SSR?
N-terminus of the nascent protein bind SSR
What happens when SRP is released?
The nascent polypeptide can enter the hydrophilic pore in the ER (Translocon)
What signal peptide ?
A sequence of N terminus amino acid that directs there Ribosomes to ER
What is a Translocon?
A pore in the ER membrane created by one or more membrane proteins
What is the major Translocon protein?
Sec61
What is there heterotimer of sec61?
Sec61a, Sec61b, Sec61g
What does post translational translocation require?
Tetramerud protein complex - Sec63 (ER membrane) and a molecular chaperone called BiP (ER lumen)
What does BiP have?
Peptide Binding domain and an ATPase domain
What do chaperones bind and stabilise?
Unfolded or partially folded proteins
What does BiP hydrolyse?
ATP and bind to polypeptide
What does binding of BiP-ADP to polypeptide prevent?
Backsliding of polypeptide out of ER
Where are all integral membrane proteins synthesised?
Rough ER
Where are plasma membrane proteins anchored to?
Rough ER during synthesis
Do plasma membrane proteins pass through the membrane into ER lumen?
No
How are the plasma membrane and proteins lodged in the ER?
Short hydrophobic sequences
How do stop-transfer anchor sequence move?
Literally between translocon subunit and become anchored in the phospholipid bilayer
Type II single pass
No N term ER signal sequence Single internal hydrophobic signal anchor sequence Bound by an SRP
Multi-pass
The protein will thread in and out of the membrane dependent on the number and position of the stop-transfer anchor sequences
What do post-translational reactions process?
Inactive, immature (precursor) proteins to mature, active products
What events occur in different proteins in a post-translational protein?
Disulfide bridges Glycosyaltion (addition of sugar residues to a protein) Protein folding Addition of Lipids Phosphorylation Partial, cont pulled proteolysis Hydroxylation Methylation/acylation/ prenylation/adenylation/acetylation Vitamin K dependent Glutamate carboxylations
Where does glycosylation commmence and progress into ?
Commences - ER Progresses - Golgi
What does the membrane-bound enzyme - oligosaccharyl transferase do?
Add 14 sugar precursor to all proteins in the rough ER
What holds the precursor oligosaccharide in the ER membrane?
dolichol
How is glycosylation trimmed?
Removal of 2X glucose and 1X mannose
What plays a critical role in the correct folding of many proteins in ER?
Re-addition of 1 glucose molecule
What helps protein fold correctly?
Chaperone resident in the rough ER lumen
What is require for correct folding of many proteins?
N-Linked Glycosylation
What are examples of chaperones?
Calnexin and calreticulin
What do calnexin do?
Bind to I completely folded proteins containing 1 terminal glucose, trapping it in the ER which is added to midfielder proteins by a glucosyltransferase
Where are disulphide bonds only formed?
Lumen of the rough ER
Where are disulphide bonds found?
Secretory proteins
What is dulsphide bond formation catalysed by?
Disulphide isomerase
Where does the immature protein accumulate?
Outer surface of the ER membrane
What does the accumulation of immature protein result in?
Formation of protrusions
What does the protrusion do?
Bud from the ER membrane
What do the bud form?
Small transport vesicles
Where does the transport vesicle move to?
Golgi complex
What does the vesicle membrane fuse to?
Golgi Membranes
What is the Golgi composed of?
Flattened spherical vesicles
What are the 3 functional regions of Golgi Complex?
Cis, medial and trans Golgi
Cis-Golgi
O-linked glycosylation
Trans-Golgi
The modifications required for sorting and targeting
How is budding initiated?
Recruitment of small GTP-Binding protein to a patch or donor membrane
How are 3 different types of coated vesicle formed?
Different small GTP binding proteins
What proteins do vesicle targeting depend on?
Rab protein and SNARE proteins
What do Rab proteins do?
Direct the vesicle to specific spots on the correct target membrane
What do SNARE proteins do?
Mediate fusion of lipid bilayers
What are the complimentary sets in which SNARE proteins exist?
V-snares - on vesicles T- snares - on target membrane
Rab1
ER and Golgi Complex
Rab2
Cis Golgi nexteork
Rab3A
Synaptic vesicles, secretory granules
Rab4/Rab11
Recycling endosomes
Rab5A
Plasma membrane, Clathrin-coated vesicles, early endosomes
Rab5C
Early endosomes
Rab6
Medial and trans Golgi cisternae
Rab7
Late endosomes
Rab8
Early endosomes
Rab9
Late endosomes. Trans Golgi network
