BMB 251 1st Edition Lecture 35 Outline of Last Lecture I. ChloroplastsII. MitochondriaIII. Types of Protein Translocationa. Gatedb. Transmembranec. VesicularIV. Nuclear Pore ComplexesV. Ran GTPaseOutline of Current Lecture VI. Mitochondriaa. Matrix spaceb. Intermembrane spacec. TOM, TIM, SAM, OXA complexesVII. Chloroplastsa. Matrix spaceb. Intermembrane spacec. Thylakoid spaceVIII. PeroxisomesIX. ERX. Single/Multipass membrane proteinsCurrent Lecture- Mitochondria and chloroplasts have double membrane organelles specializing in ATP synthesis by using electron transport and oxidative phosphorylation (mitochondria) and photosynthesis (chloroplasts)o Contain their own DNA, ribosomes and other molecules needed for protein synthesis, yet most of their proteins are made in the nucleus and imported form the cytosol- Mitochondria have two subcompartments: matrix space and intermembrane spaceo Inner membrane encloses matrix space and forms invaginations called cristaeo Outer membrane is in contact with cytosol- Mitochondria proteins are fully synthesized as mitochondrial precursor proteins in cytosol and then translocated into mitochondrial via post-translational mechanismo Many proteins entering matrix contain SS at N-terminus, which are removed by signal peptidases after import. But others made for outer membrane, inner membrane and intermembrane proteins have integral SS that is not removedThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Multisubunit protein complexes, aka protein translocators, transport protein across mitochondrial membranes o TOM complex transfers proteins across the outer membraneo Two TIM complexes transfer proteins across the inner membraneo Also SAM complex in outer membrane and OXA complex on inner membrane, which both help to properly fold and place proteins into their respective membranes- There are many interacting proteins in cytosol-many are chaperones or Hsp70 family, which make sure that precursor proteins do not inappropriately aggregate before reaching TOM- To import proteins, mitochondrial need 2 ATP hydrolysis sites (one outside the mitochondria andone inside the matrix), membrane proteins across the inner membrane via H+ gradient, and functional Hsp70o Mitochondrial Hsp70 attaches to TIM23 and pulls precursor protein into the matrix space- Outer mitochondrial membrane contains porins that allow access to inorganic ions and metaboliteso SAM helps import these to outer membrane and fold them into B-barrels- Chloroplasts have three subcompartments: matrix space, intermembrane space and thylakoid space, which is surrounded by thylakoid membraneo Protein transport here resembles that of mitochondria: both occur post-tanslationally, use separate translocation complexes in each membrane, require energy, and use amphiphilic N-terminal SS that are removed afterwardo Chloroplast use GTP and ATP hydrolysis to power import across double membrane instead of H+ gradient - Thylakoid: house protein subunits of photosynthesis system and of ATP synthaseo First proteins pass across double membrane at special contact sites to get to matrix space (known as stroma in chloroplasts) and then translocate into either thylakoid membrane or space- Peroxisomes differ from mitochondria and chloroplasts by: o Being enclosed by single membrane and not containing DNA or ribosomes  acquire most proteins from cytosol, some from ERo Major sites of oxygen utilization to carry out oxidation reactionso Generate hydrogen peroxide and contain catalase to destroy excesso Self-replicating organelleso Specific sequence of three amino acids near C-terminus serve as peroxisomal import signal o Proteins are imported from cytosol without unfolding- ER: membrane is organized into netlike labyrinth of tubules and flattened sacs (“pancakes”) that extend throughout cytosol and is continuous with outer nuclear membrane o ER and nuclear membrane share internal space, called ER lumen (or ER cisternae space)o Central role in lipid and protein biosynthesis, and serves as intracellular Ca2+ store  used for cell signaling responseso Almost all proteins destined for cell secretion are initially delivered to ER lumen o Captures proteins from cytosol as they are being synthesized. Can either be transmembrane proteins (partly translocate across ER and become embedded in it) or water-soluble proteins (fully translocated across ER membrane and released into ER lumen)- Rough ER: co-translational process, where ribosome that is synthesizing protein is attached directly to the ER membrane  one end of protein is translocated to ER already synthesized while the other end is translated there - Smooth ER: has transitional ER which contains transport vesicles containing newly synthesized proteins and lipids which bud off and go to Golgio Central in lipid metabolism - All proteins destined for ER have ER signaling sequences (SS), which takes it to ER and is cleaved off from N-terminus by signal peptidase in ER before polypeptide chain is fully synthesized o ER SS is guided to ER by two things Signal-recognition particle (SRP): travels between ER and cytosol to bind to SS SRP receptor: receives SRP in ER membraneo There are membrane –bound ribosomes on cytosolic side of ER membraneo There are also free ribosomes in the cytosol (only organelles with no membrane)- Polypeptide chains are transferred across ER membrane through a pore in a protein translocators, whose core is known as sec61 complex- In post-translocation of proteins from ER, BiP is used in junction with sec61 to pull the protein into the lumen of the ER- ER SS is read twice: once by SRP in the cytosol and once again by pore of protein translocators (this serves as start-transfer signal that opens the pore)- Single-pass membrane proteins become inserted into the ER by:o SS on N-terminal signals translocation. Hydrophobic segment provides stop-transfer signal  protein doesn’t go all the way through. SS is cleaved off, stop-transfer sequenceremains in the bilayer. The N-terminus is normally in the lumen while the C-terminus is in the cytosolo OR SS can be internal, rather than at N-terminal end. SRP binds to internal SS. From this, orientation of protein can have N-terminal in the lumen and C-terminal in the cytosol, orvice versa- Multipass membrane protein: polypeptide chain passes back and forth repeatedly across the