BMB 251 1st Edition Lecture 36Outline of Last Lecture I. Mitochondriaa. Matrix spaceb. Intermembrane spacec. TOM, TIM, SAM, OXA complexesII. Chloroplastsa. Matrix spaceb. Intermembrane spacec. Thylakoid spaceIII. PeroxisomesIV. ERV. Single/Multipass membrane proteinsOutline of Current Lecture VI. Clicker QuestionsVII. Glycosylationa. Glycoproteinsb. GlycolipidsVIII. Endoplasmic ReticulumIX. Types of Vesiclesa. Coatedb. Transportc. ClathrinX. Rab proteinsXI. SNARE proteinsCurrent Lecture- Clicker Question 1: The red portion of this protein is hydrophobic. It is flanked by charged residue as shown. + -N CThese 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.- What type of protein is this?o Type I transmembrane protein (N-terminus luminal)o Type II transmembrane protein (N-terminus cytosolic)o Type III transmembrane protein (multipass TM protein)o Cytosolic proteino None of the above **The positive charge is maintained in the cytosol- Clicker Question 2: Which of the above is an integral membrane protein? o Transmembrane domaino GPI anchoro Both o Neither- Many proteins in the ER lumen are in transit, but others are ER resident proteins (normally reside there at high concentrations) and contain ER retention signal at the C-terminus of protein that keeps it in ERo One important ER resident is protein disulfide isomerase (PDI): catalyzes oxidation of free sulfhydryl (SHI groups forms disulfide bonds (S-S) o Another important ER resident is BiP: pulls proteins post-translationally into ER via ER translocator (**BiP is a chaperone that recognizes unfolded or incorrectly folded proteins  bound BiP prevents aggregation of protein and keep it in ER- Covalent addition of sugars to proteins is one of major biosynthetic functions of ERo Half the eukaryotic proteins are glycoproteins (most come from ER because very few proteins in cytosol actually get glycosylated o Sugar (aka precursor oligosaccharides) are mostly added to NH2 groups asparagine amino acids  “asparagine-linked” or “N-linked”o Almost as soon as polypeptide chain enters ER lumen, it is glycosylatedo Less frequently (10%), glycoproteins can be made by adding sugar to –OH group on serine, threonine or hydroxylysine amino acids in Golgio Many proteins translocated to ER fail to achieve proterly folded or oligomeric state  sent back to cytosol to be degraded (aka retrotranslation or dislocation) via deglycosylation, ubiquitalation and degradation in proteasomes - Glycosylphosphatidyl-inositol (GPI) anchor: enzymes catalyze this covalent attachment to C-term of some membrane proteins destined or plasma membrane o Linkage forms in lumen of ER while transmembrane portion of protein is cleaved off o Can be used to direct plasma membrane proteins into lipid rafts or GPI’s can be released from cells in response to signals that activate phospholipase in plasma membrane- ER membrane synthesizes nearly all classes of lipids (phospholipids and cholesterol) One of the most common is phosphatidylcholine: formed in 3 steps from choline, 2 fatty acids and glycerol phosphate. Each step catalyzed by enzymes in ER facing cytosol, where all metabolites are found  phospholipid synthesis occurs mostly on cytosolic leaflet of ER. Phospholipid translocator rapidly “flip flops” lipids to lumen leaflet of ER (**process cannot be done in normal lipid bilayer) different phospholipids are equally distributed between ER leaflets- ***Proteins (cytosol)  ER  add on folding, oligosaccharides, disulfur bonds, N-linked glycosylation determines extent of protein folding- Cells continuously adjust composition of their plasma membrane in rapid response to need o Exocytosis: biosynthetic secretory pathway delivers newly synthesized proteins carbs andlipids to plasma membrane or extracellular spaceo Endocytosis: cells remove plasma membrane components and deliver them to internal compartments called endosomes  recycled to another part of plasma membrane or degraded by lysosomes **Take in important vitamins/nutrients and deliver them to cytosol - Lumen is topologically equivalent because proteins can travel through this space without having to cross a membrane  passed through compartment via transport vesicles o Transport vesicles normally bud off from one membrane and fuse with another, carrying with it membrane components and soluble molecules (cargo) o Biosynthetic-secretory pathway travels outward from ER to Golgi, with side route leadingto lysosomes o Endocytic pathway leads inward from plasma membrane o Transport vesicles are very selective- Coated vesicles: many transport vesicles bud off of membranes in a distinctive cage of proteins covering their cytosolic surface, which they discard before fusing with the target compartment (needed for two cytosolic membrane surfaces to interact directly and fuse)o Coat concentrates specific membrane proteins in specialized patch  vesicle membrane o Coat molds, forming the vesicle- Clathrin-coated vesicles: transport material from plasma membrane and between endosomal and Golgi compartmentso Clathrin is the major protein present hereo Adaptor coat binds clathrin coat to membrane and traps various transmembrane proteins, including receptors that capture cargo inside vesicle (“cargo receptors”)- Retromer: coat that forms patches dedicated to specific cargo proteins, which assembles on an endosome and forms vesicle return acid hydrolase receptors to the Golgio **Assembly of coat introduces curvature to membrane, which leads to formation of uniformly sized coated buds. Adaptor proteins bind chlathrin triskelions and membrane-bound cargo receptors  selective recruitment of both membrane and cargo molecules into vesicles. Clathrin coat is rapidly lost after vesicle forms- Soluble cytoplasmic proteins (such as dynamin) contain GTPase domains, which help vesicles (clathrin) to pinch off from donor membrane. This pinching brings two noncytosolic leaflets together and fuses them  vesicles o Hsp70 chaperone acts as an uncoating ATPase- Depending on relative efficiencies of tubule formations (Golgi) and pinching-off processes, vesicles of different sizes and shapes are produced - Specifically in finding correct target membrane is insured because all transport vesicles display surface markers that identify them according to their origin and type of