Scale in the biological world

From living cells to atoms

A cell seen by TEM

Compartmentalisation in the cell: internal membranes and the cytosol

The cytosol: more than just H2O RNAs Proteins Ribosomes

Πιθανή εξελικτική πορεία για το Ε και την πυρηνική µεµβράνη

The Origin of mitochondria: The endosymbion hypothesis

Νέα οργανίδια φτιάχνονται από προυπάρχοντα οργανίδια ιαίρεση οργανιδίων κατά την κυτταρική διαίρεση Και Ανάπτυξη των µεµβρανών µε την προµήθεια λιπιδίων και πρωτεινών ΠΡΟΒΛΗΜΑ : ΙΑΛΟΓΗ ΠΡΩΤΕΙΝΩΝ PROTEIN SORTING

The Eukaryotic Cell

Different ways to target proteins in the cell

Mitochondria are essential for life

Energy Conversion: Mitochondria and Chloroplasts Membrane-bounded bounded Occupy a major fraction of cell volume Large amount of internal membrane Common pathway for energy conversion: Chemiosmotic coupling

Chemiosmotic coupling

The Mitochondrion 1. Substantial portion of cell volume About 20% of the volume of a eukaryotic cell Mitochondrial IM is 1/3 of total cell membrane 2. Mitochondrial function supplies 30 ATP molecules (only 2 ATP from anaerobic (cytosolic) glycolysis 3. Mobile, shape-changing,fusion/separation

EM view of a Mitochondrion 3D Reconstruction mobile, shape-changing

Large GTPases control mitochondrial fusion (Fzo1, OM) fission (Dnm1, OM) and Inner membrane remodelling (Mgm1, IMS)

Mitochondrial Structure Outer Membrane (semipermeable( semipermeable) 6% of total mit.protein lipid metabolism enzymes, porin IMS 6% of total mit.protein enzymes that use ATP to phosphorylate other nucleotides Inner Membrane (impermeable) 21% of total mit.protein ATP synthase,, respiratory chain enzymes, transport proteins Matrix 67% of total mit.protein Hundreds of enzymes, DNA, ribosomes, trnas

Mitochondrial Energy Metabolism

The Electrochemical Proton Gradient 140 mv 60 mv (-1 ph unit) TOTAL 200 mv

Active transport processes are driven by the electrochemical proton gradient

The Respiratory chain consists of 3 large membrane-embedded enzyme complexes

ATP Synthase is a reversible coupling device: It interconverts the energies of the electrochemical proton gradient and chemical bonds

Functional Complexity Structural Complexity Respiration and ATP Synthesis Synthesis of heme, lipids, amino acids and nucleotides Intracellular homeostasis of inorganic ions 5-15% of total cell protein 20% volume of eukaryotic cell IM is 1/3 of total cell membrane About 1000 different polypeptides (600 in yeast) Only a dozen encoded by mtdna

Protein import is the major mechanism of mitochondria biogenesis

Identification of components of the Mitochondrial Protein Import System Genetic analyses (fungal genetics) In vitro import assay system with isolated functional mitochondria Chemical Crosslinking Biochemical reconstitution

Import into the matrix - 1 Depends on a matrix-targeting signal: The presequence Cleavable, usually located at the N-terminus usually 12-15 residues long amphiphilic, with positively charged residues on one side of an a-helix

Presequence binding to Tom20

Import into the matrix - 2 This is a multistep process Interactions with chaperones in the cytosol keep the precursor in an unfolded conformation ( import-competent ) Different import complexes in the OM (TOM complex) and the IM (TIM complex). Electrostatic interactions between the positive presequence and negative patches of receptors along the import pathway: The Acid chain hypothesis- Gradation of affinities leads the presequence along the import pathway The electrophoretic function of the potential across the IM draws the precursor across the IM The pulling force of the translocation motor mhsp70/tim44 actively draws the precursor to complete translocation

Import into the matrix - 3 Energy requirements: ATP Hydrolysis In the cytosol (function of ATPase chaperones) In the mitochondrial matrix (Hsp70 translocation motor) Electrochemical potential across the inner membrane

Import into the matrix - 4 Components TOM Complex: Receptors: Tom70, Tom20, Tom37, Tom22 Channel-forming: Tom40, Tom5 Channel modulating: Tom6, Tom7 TIM Complex: Receptor: Tim23 Channel-forming: Tim23, Tim17 Translocation motor: Tim44, Hsp70, GrpE (co-chaperone) chaperone)

Protein import into the matrix (70% of mitochondrial proteins) The essentials: presequence (amphipathic, positively charged) energetics: electrostatic and hydrophobic interactions of the presequence ATP hydrolysis (cytosolic chaperones and mthsp70) electrophoretic function of the electrochemical potential

Summary of Protein Import into the Mitochondrial Matrix II I IV III

Import into the IMS A. Variation of the matrix targeting pathway example: cytochrome b2 B. Distinct pathway involving a specific IMS targeting signal example: mitochondrial heme lyases

Cytb2 IMS targeting - 1 The Signal: Bipartite nature: matrix targeting signal followed by an IMS sorting signal IMS sorting signal contains mainly uncharged residues cleaved by specific IMS protease NOT very highly conserved

Cytb2 IMS targeting - 2 Energetics: Electrochemical potential absolutely essential but ATP hydrolysis s NOT required Components: Known Subunits of the TOM complex TIM23 complex IMS sorting peptidase Mechanism: Stop-transfer mechanism: the hydrophobic sorting signal is stuck at the TIM23 complex and laterally diffuses out into the lipid bilayer of the IM

Import into the IMS A. Variation of the matrix targeting pathway example: cytochrome b2 B. Distinct pathway involving a specific IMS targeting signal example: mitochondrial heme lyases

The Signal: Internal Heme Lyase IMS targeting - 1 about 60 residues long highly conserved sequence highly hydrophilic (30% charged residues, similar number of + and - charged residues, distributed throughout the sequence) mainly a-helical, a but NOT amphiphilic Key reference: Diekert et al Proc. National Acad. Sci.. USA 1999, 96, 11752-11757 11757

Energetics: Heme Lyase IMS targeting - 2 Energy is provided by high affinity interactions with import components in the OM (TOM complex) and the IMS (trans side of Tom40 and unknown components) Components: Known Subunits of the TOM complex others unidentified yet (IMS?) Mechanism: Still unknown but TOM subunits thought to play a major role

Import into the IM A. Some IM proteins contain a presequence-like region internally: They are targeted via the TIM23 complex following a variation of the matrix targeting pathway The presequence is decoded by the TIM23 complex in a looped conformation B. IM proteins without a presequence: Example: Mitochondrial carriers like the ADP/ATP carrier (AAC) They follow a distinct import pathway

In mammalian cells mitochondrial carriers are involved in a number of disorders: diseases, like several myopathies obesity programmed cell death (apoptosis)

The Mitochondrial Carrier Family Function as metabolite transporters 37 proteins in yeast (10-15% of the total mitochondrial protein) 30-35 kda Common topology: 3 similar repeated motifs (3X2 helix model)

Active transport processes are driven by the electrochemical proton gradient

Summary of Protein Import into the Mitochondrial Matrix II I IV III

II I III IV V

Properties of the small Tim proteins - 1 Sequence characteristics: They are all homologous They are intrinsically soluble (No transmembrane domains) They have a putative zinc-binding motif, twin CX3C motif X 15/16 Found in all eukaryotic cells Human homologue of Tim8 H 2 N C X 3 C Zn 2+ C X 3 C COOH involved in Mohr-Tranebjaerg syndrome Organisation in assemblies: Tim9/10 and Tim8/13 are found in 70 kda complexes in the IMS Tim12 is associated with the membrane-embedded embedded TIM22 complex (IM)

Properties of the small Tim proteins - 2 Function: Tim9/10 (essential( essential) ) function as chaperones in the IMS and facilitate targeting to the IM Tim12 (essential( essential) ) facilitates insertion in the context of the TIM22 complex Tim8/13 are non-essential but also seem to function as chaperones in the IMS

Distinct features of the carrier pathway compared to matrix targeting : 1. Internal targeting signals spanning the whole protein 2. Requirement for a chaperone function in the IMS 2. No ATP hydrolysis in the matrix is necessary 3. No translocation motor in the matrix is required 4. The electrochemical membrane potential across the IM is enough to complete insertion at the IM

KEY POINTS 1. Multiplicity of pathways to cope with targeting and sorting to the different mitochondrial compartment 2. All mitochondrial import processes are mediated by specific translocation machineries in all subcompartments 3. Only one TOM complex in the outer membrane through which all precursors are imported. After the barrier of the outer membrane, import routes to subcompartments divert. At least two distinct TIM complexes in the inner membrane

TOM SAM TIM23 TIM22 OXA1

FUNCTIONAL and STRUCTURAL similarities among Mitochondria and Chloroplasts

Chloroplast features Photosynthesis: light-driven ATP synthesis Member of the plastid family of organelles: unique to plant cells All contain their own DNA

Chloroplasts resemble mitochondria but have an extra compartment

Comparison of a Mitochondrion and a Chloroplast

Photosynthesis reactions in a chloroplast Light reactions: Light-driven production of ATP and NADPH Dark reactions: Conversion of CO2 to carbohydrate Carbon-Fixation is catalysed in the chloroplast stroma by ribulose biphosphate carboxylase (500 kda), 50% of total chloroplast protein and the most abundant protein on earth

Electron Flow during photosynthesis in the chloroplast thylakoid membrane

The Electrochemical Proton Gradient is similar in mitochondria and chloroplasts

Overview of protein import into chloroplasts

Toc GTP GTP P 159 ATP P 14-3-3 P hsp70 cytosol 34/33 75 64 OE Tic 62 40 110 hsp70 22 20 55 intermembrane space IE hsp100 hsp70 stroma SPP hsp70

Targeting of thylakoid proteins At least 4 different pathways: A. Soluble thylakoid lumen proteins 1. Sec pathway (unfolded proteins) 2. Tat pathway (fully-folded folded proteins, ph- dependent) B. Hydrophobic membrane proteins 1. SRP pathway 2. Spontaneous insertion

Toc Tic envelope Spontaneous insertion Sec/ATP A TAT/ ph 43 54 43 FtsY SRP stroma YE B C A/E Alb3 lumen