Endoplasmic Reticulum (ER) and Golgi Apparatus PDF

Endoplasmic Reticulum (ER) and Golgi Apparatus Dr Wendy Francis [email protected] Learning objectives Describe the endomembrane system Describe the structure and function of the smooth and rough endoplasmic reticulum Explain the basic transport mechani...

Endoplasmic Reticulum (ER) and Golgi Apparatus Dr Wendy Francis [email protected] Learning objectives Describe the endomembrane system Describe the structure and function of the smooth and rough endoplasmic reticulum Explain the basic transport mechanisms between the endoplasmic reticulum and Golgi apparatus Describe the structure and function of the Golgi apparatus Reading List Pgs 174-178 Chapter 12 Endoplasmic reticulum Endomembrane system Multitude of membrane-enclosed organelles Nuclear envelope Endoplasmic reticulum (ER) Golgi apparatus Lysomes, vesicles Plasma membrane Compartmentalises organelles Complex distribution systems Membranes are not identical and are dynamic in nature  Endoplasmic Reticulum (ER) Membrane constitutes more than half of the total membrane of an average animal cell Labyrinth of branching tubules and sacs which interconnect, continuous with outer nuclear membrane Single internal space called ER lumen Endoplasmic Reticulum (ER) Rough ER Flattened sheets with ribosomes bound to cytosolic surface Smooth ER Tubular structure with no ribosomes  ER Function Smooth ER (diverse metabolic functions) Lipid biosynthesis (e.g. oils, steroids, phospholipids) Carbohydrate metabolism Calcium storage Detoxification of drugs and poisons Rough ER Protein biosynthesis  Water soluble secretory proteins  Transmembrane proteins Protein folding and modification Removal of incorrect proteins  Smooth ER: Synthesis of lipids & steroid hormones Each step in lipid synthesis is catalysed by enzymes within the SER What are membranes largely made up of? Phospholipids Glycolipids ER (5%) membran Cholesterol e Cholesterol: role in membrane structure and acts to reduce permeability of cell membrane Involved in bile synthesis and steroid synthesis  Smooth ER Calcium storage Ca2+ pump transports Ca2+ from cytosol into ER lumen where it’s stored at Ca2+ binding proteins Specialised sites e.g. sarcoplasmic reticulum in muscle to allow for contraction (release of Ca2+) and relaxation of muscle (Ca2+ uptake) Drug detoxification Site of specialist enzymes that catalyse a series of reactions to detoxify lipid-soluble drugs and harmful metabolites produced during metabolism Example: cytochrome p450 enzymes in liver hepatocytes Addition of hydroxyl groups to drug molecules, making them more water soluble and easier to excrete from the body  Rough ER: protein biosynthesis Ribosomal sub-units complex together with mRNA in the cytoplasm to initiate protein synthesis N terminus Amino Acid Signal Sequence N-terminal or internal Localisation signal for the protein  Rough ER: protein biosynthesis Translation always begins in the cytosol (except for a few proteins made in mitochondria or chloroplasts) Khan Academy  Protein Distribution NO Amino Amino Acid Acid Signal Signal Sequence Sequence Protein remains within the cytosol Protein distribution to the ER Step 1 Signal recognition particle (SRP) recognises and binds to the signal sequence. Step 2 SRP-ribosome complex binds to SRP receptor and the translocation complex on ER membrane  Protein distribution to the ER Step 3 The signal sequence enters the translocon in ER membrane Step 4 Polypeptide chain is synthesised and translocated across the Step 5 membrane For secretory proteins, the protein is completely translocated across the membrane. The signal sequence is cleaved by a signal peptidase and protein released  Transmembrane Proteins Type I: single pass N terminus signal sequence targeted N terminus signal sequence to ER lumen 2nd hydrophobic region in the protein called a ‘Stop Transfer Signal’, which closes the translocon The transmembrane protein moves along the membrane laterally The amino acid signal sequence is cleaved  Transmembrane Proteins Types II/III: single pass Internal amino acid signal sequence Internal signal sequence Internal signal sequences can be orientated through the translocon either way. Either the carboxy or amino terminus can be located in the cytosol The transmembrane protein moves along the membrane laterally using the signal sequence Examples: tyrosine kinase receptors, integrins or cytokine receptors  Transmembrane Proteins Internal signal Multipass transmembrane sequence Alternating internal Signal Sequences and Stop Transfer Signals Internal signal sequences can be orientated through the translocon either way. Either the carboxy or amino terminus can be located in the cytosol Each time a hydrophobic section enters the translocon the protein moves laterally along membrane https://www.nature.com/scitable/topicpage/gpcr- 14047471/ https://www.youtube.com/watch?v=u0g82N ul1Qc  Protein folding ER is a major protein folding site Molecular chaperones and folding enzymes in ER lumen assist in folding and control subsequent release from the ER  Protein folding by ER chaperones BiP (Binding immunoglobulin protein) chaperone protein Binds to polypeptide chain ATP-driven (binding and release): drives unidirectional translocation Mediates protein folding Prevents protein aggregation Detects incorrectly folded proteins by binding to exposed amino acids that shouldn’t be exposed Prevents incorrectly folded proteins leaving ER  Protein folding: disulfide bond formation Resident proteins in ER lumen contain ER retention signal (at C terminus) Protein disulfide isomerase: catalyses oxidation of sulfhydryl groups on cysteines to form disulfide (-S-S-) bonds  N-linked glycosylation About half of the proteins processed in ER are glycoproteins. 90% glycoproteins undergo N- linked glycosylation in ER lumen Addition of a pre-formed precursor oligosaccharide (14 sugars) onto side chain NH2 group of asparagine.  N-linked glycosylation: Why Stabilises proteins by masking hydrophobic stretches, proteolytic cleavage sites or immune recognition: prevents early degradation Increase protein solubility and prevents aggregation Facilitates protein folding Acts as a critical check point in glycoprotein folding An unfolded protein will undergo continuous cycles of glucose trimming and glucose addition until it has achieved its fully folded state Chaperone proteins recognise and bind to glucose molecules Once 3 glucose and 1 mannose trimmed, the protein can be transported to the Golgi  Endoplasmic reticulum quality control (ERQC) Very inefficient process with more than 80% of proteins never achieving their fully folded state The accumulation of unfolded, misfolded or damaged proteins leads to ER stress, which is dealt with through protein degradation Ubiquitin-proteosome system: translocated into the cytosol, tagged with ubiquitin, and degraded by proteosomes Autophagic-lysosomal system: aberrant protein fragments degraded by lysosomes When the misfolded proteins exceed ER degradation capacity, the unfolded protein response is activated to eliminate misfolded proteins and reduce the synthesis Jiang etof al new proteins (2021) in Reticulum Endoplasmic the ER Quality Control in Immune Cells. Front  ER Summary ER part of the endomembrane system continuous with nuclear envelope Factory producing almost all the cell’s proteins and lipids Rough ER primarily responsible for protein biosynthesis: secretory or transmembrane Smooth ER has diverse metabolic functions including lipid biosynthesis, carbohydrate metabolism, calcium storage and drug detoxification Signal sequence and signal-recognition particle directs ribosome and polypeptide chain to ER membrane Chaperone proteins and enzymes in ER lumen, mediate protein folding N-linked glycosylation – signalling molecule in the folding process  Transport mechanisms Endocytosis: Internalisation of nutrients or removal of plasma membrane components Exocytosis: Secretory pathway outwards from the cell Transport vesicles: Membrane enclosed transport containers to move cargo between compartments  Transport vesicles 3 main types of coated vesicles: 1) Clathrin-coated: mediate transport from PM and between endosomal and trans Golgi network 2) COPI-coated: transport at Golgi network (retrieval pathway) 3) COPII-coated: transport from ER to Golgi  Transport vesicles COPII COPI clathrin coat coat coat ER Exit sites Forward and Endosomal concentrate reverse movement movement of  Vesicular tubular clusters Fusion of ER derived COPII vesicles into Vesicular Tubular clusters Move along microtubules towards Golgi Apparatus COPI coated transport vesicles then bud off the vesicular tubular clusters (ER retrieval signals to return ER resident proteins to ER) Golgi Apparatus https://www.youtube.com/watch?v=rvfvRgk0MfA  Golgi Apparatus Multiple discrete compartments (cisternae) Cis Golgi network (collection of fused vesicular tubular clusters arriving from ER): modification of proteins, lipids and polysaccharides begins Golgi stack: further modification Trans Golgi network: Sorting and distribution centre Oligosaccharide processing in Golgi Series of enzymes within each cisternae perform different functions in removal or addition of sugars Golgi Apparatus Function Complete the processing of proteins and lipids received from the ER Packaging and transport to eventual destinations (POST OFFICE!)  Summary Correctly folded proteins in ER congregate at exit sites and are packaged into COPII coated transport vesicles Vesicles shed their coat and fuse with each other to form vesicular tubular clusters Clusters move on microtubule tracks to Golgi apparatus to form cis Golgi network Movement of cargo from cis Golgi network to the trans Golgi network Lipids and proteins are modified within each cisternae of the Golgi apparatus Protein sorting and distribution in trans Golgi network