Lecture 13 / 14

Question 1

mitochondria

Answer 1

energy production derived from carbohydrate & lipid catabolism (via oxidative phosphorylation [TCA cycle])

Question 2

mitochondria often forms…

Answer 2

mitochondria often form ‘mitochondrial network’ - highly branched, long & interconnected series of tubules

Question 3

__allows for cell-wide co-ordination of organelle’s functioning and biogenesis

Answer 3

mitochondrial network’ allows for cell-wide co-ordination of organelle’s functioning and biogenesis

Question 4

chloroplasts

Answer 4

– energy production and carbohydrate synthesis via photosynthesis

Question 5

mitochondrial tubules are mobile and undergo …

Answer 5

fusion and fission

Question 6

Mitochondrial fission

three-step process:

Answer 6

Step1
• ER tubules encircle mitochondrion (via change in ER shape) at future fission site and in itiate constriction = ER subdomain MAM

Step 2
* Drp1 recruited from cytoplasm to constriction site and assemble into helices (DRP1 ‘ring’) around surface of mitochondrial outer membrane
* Drp1 = Dynamin-related protein 1 - member of dynamin GTP-binding protein family responsible for scission of other cellular membranes (e.g., clathrin vesicles @ TGN or pm)
* recruitment of Drp1 (and ER tubule) mediated by lipid ‘microdomain’ in mitochondrial outer membrane enriched in cardiolipin - mitochondrial-specific membrane phospholipid normally only found in inner membrane - recruited from inner to outer membrane at constriction site
* Drp1 interacts with cardiolipin at constriction site

Step 3
* conformational change in Drp1 ‘ring’ due to GTP hydrolysis results in further membrane constriction and fission – formation of two ‘daughter’ mitochondrion

Question 7

__ interacts with __ at constriction site

Answer 7

Drp1 interacts with cardiolipin at constriction site

Question 8

Phagocytosis

Answer 8

uptake of large materials (bacteria, yeast, etc) from the extracellular space by specialized cells (e.g., macrophages)

Question 9

Endocytosis

Answer 9

bulk-phase endocytosis (pinocytosis) & receptor-mediated endocytosis

Question 10

Mitochondrial fusion

often occurs in response to..

Answer 10

• cell stress - increased need for coordinated functioning of mitochondria ‘network’
• requires coordinated (sequential) fusion of both mitochondrial membranes
  first outer membranes fuse; then inner membranes fuse
  order of fusion essential to maintain mitochondrial (sub)compartmentalization (‘homeostasis’)

Question 11

Mitochondrial fusion

3-step process - involves various mitochondrial membrane proteins, energy (GTP), and remodeling of mitochondrial membrane phospholipids

Answer 11

Step 1: outer membrane ‘tethering’
* GTPase mitofusins - Mfn1 and Mfn2
* Mfn1 and Mfn2 are integral outer membrane proteins which possess cytoplasmic-facing GTPase domain and long, coiled-coil, protein-protein interaction domain
* located on adjacent mitochondria - link together in GTP-dependent manner to form ‘organelle tethering complex’
* proper Mfn1/2 binding (e.g., prevention of ‘self’ binding) regulated by other mitochondrial outer membrane proteins - Bak and Bax

Step 2: outer membrane fusion
* formation of outer membrane lipid ‘microdomains’ at sites of Mfn1/2 ‘tethering’
* Phospholipase D converts cardiolipin (moves from inner to outer membrane) into phosphatidic acid (‘cone-shaped lipid’) causing the outer membrane to curve inward and promotes Mfn1/2-mediated membrane fusion (via GTP hydrolysis)

Step 3: inner membrane (cristae) fusion
* inner membrane fusion mediated by OPA1 (OPtic Atrophy 1) integral inner membrane-bound mitofusin (≈ Mfn1/2) containing intermembrane space-facing GTPase domain
* OPA1 proteins on adjacent inner membranes interact in GTP-dependent manner to promote membrane fusion (via GTP hydrolysis)
* OPA1 binding regulated by other mitochondrial inner membrane proteins
e.g., Prohibitin - ensures that OPA1-mediated fusion occurs only between ‘different’ inner membranes prevents ‘self’ fusion of cristae within same mitochondrion

Question 12

Majority of mitochondrial proteins are …

Answer 12

(soluble & membrane-bound) nuclear encoded, synthesized on ‘free’ ribosomes in cytoplasm, and targeted post-translationally (i.e., after translation) to organelle

Question 13

all nuclear-encoded mitochondrial proteins possess unique ____ serving as ‘_____’ to mediate ___ and to ___

Answer 13

all nuclear-encoded mitochondrial proteins possess unique targeting sequences serving as ‘zipcodes’ to mediate protein targeting from cytoplasm to surface of mitochondrion and to 1 of 4 specific mitochondrial subcompartments

Question 14

Targeting and import of mitochondrial matrix proteins

Answer 14

Matrix-destined proteins typically contain a 20-50 amino acid-long matrix targeting sequence situated at the N terminus. This sequence comprises an amphipathic alpha helix with positively-charged residues (R/K) on one side and hydroxylated residues (S/T) on the other. It is cleaved upon protein import into the matrix. This sequence is crucial for directing nascent matrix proteins to the cytoplasmic surface of the mitochondrion and facilitating their translocation across both the outer and inner membranes.

Question 15

What does it mean to consists of amphipathic a-helix

Answer 15

Protein is enriched in positively-charged residues (R/K) on one side of helix and hydroxylated (S/T) residues on other side

Question 16

Matrix protein targeting & import

• in cytoplasm, precursor protein is synthesized on free ribosomes and recognized by cytoplasmic molecular chaperones

Answer 16

Step 1
Mitochondrial mRNA localization involves the concentration of mRNAs encoding mitochondrial proteins in the cytoplasm surrounding mitochondria, forming a ‘mitochondrial RNA cloud.’ This localization is facilitated by RNA-binding proteins on the outer mitochondrial surface that interact with unique sequences in mitochondrial mRNA UTRs, acting as anchors. As a result, protein synthesis occurs adjacent to the mitochondrial surface, allowing for spatial control of mitochondrial protein gene expression and efficient post-translational targeting of proteins to the mitochondria.

Step 2
* at surface of mitochondrion, protein’s matrix- targeting sequence recognized (bound) by import receptor complex
* consist primarily of two integral outer membrane proteins - Tom20 and Tom22 - consisting of several accessory proteins
* serve as ‘scaffold’ to mediate subsequent precursor protein transfer from import receptor complex to general import pore

Step 3
* precursor protein passed from import receptor to general import pore in outer membrane
* consists primarily of integral outer membrane protein Tom40 - referred to as “general” import pore
* all (most) mitochondrial proteins (both matrix and membrane-bound) access mitochondria initially through TOM40

• Tom40 forms transmembrane channel
• allows for protein translocation across (or into, for membrane proteins) the outer membrane

Steps 4 and 5
* precursor protein transferred through general import pore and then through adjacent inner membrane channel
* consists of integral inner membrane proteins Tim17, Tim23, and TIM44

• general import pore and inner membrane channel adjacent to each other at contact sites
• places where outer and inner membranes are closely appressed – intermembrane space reduced or absent at contact sites
• contact sites maintained by interactions of Tom40 and Tim23/17 intermembrane-space-facing domains
• precursor protein translocation occurs across both membranes sequentially
• N-terminal matrix-targeting sequence of precursor protein exits inner membrane channel into matrix
• matrix-targeting sequence cleaved by matrix processing protease
• emerging precursor protein also recognized and bound by matrix Hsp70
• located at matrix-face of inner membrane channel via binding to Tim44
• inner membrane channel accessory protein
• matrix Hsp70 acts as molecular motor (‘ratchet’)
• Tim44-bound Hsp70 undergoes ATP-dependent conformational changes that ‘pulls’ protein into matrix AND prevents ‘back sliding’ of protein back into cytoplasm

Steps 6 and 7 final steps…
* imported, cleaved (mature) protein in matrix folds without further assistance into final, active conformation [Step 6]
OR
* final folding of imported, cleaved protein in matrix requires additional matrix-localized molecular chaperones and ATP hydrolysis [Step 7] represents an additional (4th) requirement for energy input in mitochondrial matrix protein import

Question 17

at surface of mitochondrion, protein’s matrix- targeting sequence recognized (bound) by import receptor complex

• consist primarily of two integral outer membrane proteins ___

Answer 17

Tom20 and Tom22

Question 18

Tom20 and Tom22 serve as an___

Answer 18

Scaffold

Question 19

precursor protein passed from import receptor to general import pore in outer membrane

• consists primarily of integral outer membrane protein___

Answer 19

Tom40 - referred to as “general” import pore

Question 20

Tom40 forms …

Answer 20

Tom40 forms transmembrane channel
• allows for protein translocation across (or into, for membrane proteins) the outer membrane

Question 21

precursor protein transferred through general import pore and then through adjacent inner membrane channel

• consists of integral inner membrane proteins

Answer 21

Tim17, Tim23, and TIM44

Question 22

Contact sites

Answer 22

general import pore and inner membrane channel adjacent to each other at contact sites
• places where outer and inner membranes are closely appressed – intermembrane space reduced or absent at contact sites
• contact sites maintained by interactions of Tom40 and Tim23/17 intermembrane-space-facing domains
• precursor protein translocation occurs across both membranes sequentially

Question 23

matrix-targeting sequence cleaved by

Answer 23

matrix processing protease

Question 24

emerging precursor protein also recognized and bound by …

Answer 24

matrix Hsp70
- located at matrix-face of inner membrane channel via binding to Tim44
- inner membrane channel accessory protein

Question 25

What are the two final steps of Matrix protein targeting & import

Answer 25

Steps 6 and 7 final steps…

• imported, cleaved (mature) protein in matrix folds without further assistance into final, active conformation [Step 6]

OR

• final folding of imported, cleaved protein in matrix requires additional matrix-localized molecular chaperones and ATP hydrolysis [Step 7] represents an additional (4th) requirement for energy input in mitochondrial matrix protein import

Question 26

What is required for cytosolic Hsp70 to maintain bound precursor protein in a partially unfolded, import- competent state

Answer 26

in cytoplasm, ATP hydrolysis required for cytosolic Hsp70 to maintain bound precursor protein in a partially unfolded, import- competent state

Question 27

during protein translocation [Steps 4-5], import driven partially by H+ electrochemical gradient across inner membrane – established during electron
transport

H+ concentration in intermembrane space vs matrix

Answer 27

[H+] intermembrane space > [H+] matrix

positively-charged residues in amphipathic matrix- targeting sequence are attracted (‘pulled’) to less positively-charged matrix