05B Protein Transport [ Mitochondria - Peroxisome ]

What are the delivery sites for mitochondria

Outer membrane

Intermembrane space

Inner membrane

Mitochondrial matrix

What is the required conformation of your proteins when they get transported to the mitochondrial matrix

Linear

General requirements for the transfer into the mitochondrial MATRIX

signal sequence : N - terminal MTS ( mitochondrial targeting sequence ) rich in SKL

Energy - ATP [ pathway 2 ]

Receptor : TOM Receptor

Translocator : TOM complex translocation channel and receptors; OM

When does cleaving occur in mitochondrial transport

Cleaving occurs depending on whether the protein is either inserted or released

Cleaved by peptidases : inside mitochondrial matrix, N-terminal MTS

NOT cleaved: those with internal sequences or internal MTS

Process of matrix protein transport

1. Hsp70 binds to your polypeptide (detaches by hydrolysis of ATP as it moves through TOM)

2. transit seq binds to TOM complex on the outer mitochondrial membrane

3. Transit peptidase cleavage enzyme to remove the signal peptide

4. Polypeptide moves through TOM and TIM

5. Mitochondrial Hsp70 Chaperones via ATP Hydrolysis

6. Polypeptide folds via atp and hsp60

These molecules that binds to the polypeptide in the cytosol before protein transport into the mitochondrial matrix prevents the early folding of the polypeptide and ensures proper folding at the same time

Cytosolic Hsp70 ( if mitochondrial → Mitochondrial hsp70, Hsp60 (for proper protein folding) and Hsp10 )

Translocase of the inner membrane

TOM AND TIM

Translocase complex that helps in the proper folding of the protein in the outer membrane of the mitochondria, assisting TOM Complex

SAM Complex

This translocase mediates the insertion of the linear membrane proteis which are synthesized within the mitochondria (bec. There are ribosomes in the mitochondria )

Oxa complex

translocation in inner mitochondrial membrane That Contains import ATPase. It specifically pulls proteins across the inner mitochondrial membrane, across the matrix

TIM 23 complex

Transport complex located in the inner membrane of the mitochondria (w/out atpase )

TIM22 complex

INTERMEMBRANE PROTEIN TRANSPORT Requirements

Signal Sequence: Intermembrane space-targeting sequence

Translocation machinery: Translocase of the outer membrane (TOM) and the TIM complexes.

Receptor:

Energy:

INTERMEMBRANE PROTEIN TRANSPORT explain

1. your polypeptide contained MTS and intermembrane space targeting sequence

2. Once the intermembrane space passes through the TIM, IT GETS STUCK and it laterally diffuses in the inner membrane space

3. Protease would then cleave the intermembrane space targeting sequence to free the polypeptide to the intermembrane space

INNER MEMBRANE PROTEIN TRANSPORT three paths

Contains stop-transfer sequences

A - [stop-transfer sequences] stops entering and diffuses laterally → becomes incorporated into the inner membrane.

B - [ Oxa1-targeting sequence ] continues through TIM and enters the matrix. the sequence would just interact with OXA 1 that would allow it to stay in the inner membrane

C - [ Internal targeting sequences ] - polypeptide is retained in the inner membrane. As any of these sequences pass through, they laterally diffuse and gets embedded

What are the three energy sources for mitochondrial transport

Pathway 1: Spontaneous

Pathway 2 and 3 : ATP and membrane potential

DELIVERY SITES For chloroplast

o Outer membrane

o Intermembrane Space

o Inner membrane

o Mitochondrial matrix

o Stroma

o Thylakoid Membrane

o Thylakoid Lumen

There are two signal sequences that is observed in the chloroplast

Chloroplast transit peptide ( CTP )

Thylakoid transport signal ( TTS )

This signal sequence of chloroplast are N-Terminal peptides that have variety of amino acid sequences.

Chloroplast transit peptide

An observable ___ moiety is conserved at every second residue of CTP

Alanine

When are Thylakoid transport signals used

observed when CTP is cleaved and proteins are transported to the thylakoid membrane, and finally to the thylakoid lumen

What enzyme do you use to cleave Chloroplast Transport peptide, how about the thylakoid transport signal

stromal processing peptidase before it folds

thylakoid processing peptidase

Chloroplast transport requirements

Sequences: CTP and TTS

translocators : TIC TOC

Receptors

Energy : stroma transport → ATP/GTP, thylakoid transport → facilitated by several independent precursor-specific pathways

Sources of energy for transport to the thylakoid.

o Sec pathway [ ATP PMF ]

o SRP-like pathway [ ATP PMF ]

o TAT pathway [ PMF ]

o Spontaneous insertion

complex facilitates the entry of cytosolic proteins to the chloroplasts; located in the OM

Toc

complex facilitates the entry of proteins to the stroma, located in the IM

Tic

This pathway of Sources of energy for transport to the thylakoid uses atp and pmf and creates a sec homologue protein

Sec pathway

This pathway of Sources of energy for transport to the thylakoid uses atp and pmf and creates a signal recognition particle

Srp like pathway

This pathway of Sources of energy for transport to the thylakoid uses pmf only that has seq. requiring to contain 2 arginines

Twin arginine translocation TAT pathway

Process of chloroplast transport

Peptide has ctp and tts, ctp binds to the receptor protein in the toc complex

Enters the tic complex ( translocation is atp ro gtp dependent )

Cleavage of ctp by stromal processing peptidase

Four routes to translocate protein into the thylakoid space

Cleavage of tts by thylakoid processing peptidase

Mature protein is now in the thylakoid space

Why is the Transport of peroxisomal proteins is very conspicuous

Peroxisomal proteins must first fold into mature conformations

They must also pre-associate with cofactors before import from the cytosol

Peroxisome two localization areas

peroxisomal membrane

peroxisomal lumen or matrix

What do you call the core of the peroxisome

Urate oxidase crystalline core

Two targeting sequence of peroxisomes

C-terminal SKL targeting sequence (Ser-Lys-Leu) [ note cleaved due to binding to PTS1R ]

N-terminal uptake-targeting sequence [ cleaved ]

This signal sequence of peroxisomes is seen in catalase, fatty acyl CoA oxidase in rat liver, urate oxidase in cucumber, and firefly Luciferase

C-terminal SKL targeting sequence (Ser-Lys-Leu)

Explain the experiment on universality of peroxisome-targeting mechanism.

Ok

Why is cleaving absent in C-Terminal SKL targeting sequence

As the PTS1R that it binds to acts dually as a functional sequence and as recognition sites which direct the insertion of these proteins into the peroxisome.

Proteins with N-terminal targeting sequence in peroxisomes binds to what receptors

cytosolic receptor protein called PTS2R, which directs the precursor protein to a

receptor on the peroxisomal membrane.

The peroxisomal membrane protein lacks this sequence

SKL sequence.