Lecture 11 - Protein Sorting Flashcards by Jihan Haji

What are the 3 mechanisms that proteins can move from one compartment to another?

Gated transport Transmembrane transport Vesicular transport

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What is gated transport?

Movement between cytosol and nucleus through nuclear pore complex which act as a selective gate that actively transport certain molecules

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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

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What is vesicular transport?

Membrane-enclosed transport packages. Move between different compartments (e.g. ER to Golgi)

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Why does targeting occur?

Information needed for the process is embedded in the structure of these proteins: signal sequence

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How is the information encoded?

Primary structure (amino acid sequence)

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How are targeting information be generated?

Modification to a protein in post-translational maturation event - processing

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What removes signal sequence?

Specialised signal peptidase

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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

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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

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When do Ribosomes bind to the ER membrane?

Co-translational translocation

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What happens when Ribosomes complete synthesis of protein?

Release it prior to post-translational translocation

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Approximately how many peptide bonds are synthesised in free Ribosomes in the cytosol?

70 peptide bonds

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What directs the Ribosomes to the ER?

A sequence of N-terminus amino acids

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What does the signal peptide allow?

Free Ribosomes to become membrane associated and growing polypeptide to cross the ER

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What bridges between the ribosome and the ER membrane that enhance ribosome Binding to the ER?

Mg2+ ions

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What part of the protein is highly hydrophobic?

N-terminal 40 residues protrude from the 60S ribosomal subunit

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What does signal recognition particle (srp) bind to?

Ribosome and signal peptide

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What is necessary for SRP Binding?

The unfolded state of the nascent peptide chain

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What does SRP do?

Interacts with ribosome, required for protein

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What does SRP contain?

6 discrete polypeptides 7S RNA 300 nucleotides long

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What is RNA related to?

Highly repetitive Alu DNA

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What are the SRP proteins in S-domain?

S19, S54, S68, S72

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What are the SRP proteins in Alu-domain?

S9 and S14

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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?

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