Cytoplasmic Membrane Systems II Lecture 12

How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur! (Discuss with Lecture 13)

The Endomembrane System Dynamic, integrated, ordered and membrane bound system Components: Nuclear Envelope Endoplasmic Reticulum Golgi Apparatus Lysosomes EndosomesVesicles Function in: 1)Regulating Protein Trafficking- Processing, Sorting and Transporting 2) Performing Various Metabolic Functions in the Cell

The Endomembrane System 1.Transmembrane Transport: Targeting Proteins to the ER 2.Vesicular Transport: Sorting and Transport of proteins Through the Endomembrane System

Components of Endomembrane System are Connected Either through Direct Contact or by Transfer of Membrane Bound Vesicles Exocytosis Endocytosis

The Endoplasmic Reticulum is Morphologically and Functionally Subdivided Three contiguous ER membrane domains: Rough ER- System of Interconnected Flattened Sacs With Attached Ribosomes Proteins, lipids and carbohydrates synthesis; assembly and folding of proteins Transitory ER- Where vesicles exit the ER to the Golgi Smooth ER- System of Interconnected Tubules without Ribosomes Attached Synthesis of Lipids for Membranes: Phospholipids and Cholesterol

Functions of the Rough ER A major site of the synthesis of proteins, lipids and carbohydrates; assembly and folding of proteins Proteins produced include: membrane, secretory, lysosomal Best studied in cells that secrete large quantities of proteins (pancreatic enzymes; mucus-secreting cells lining the digestive tract)

The Rough ER is Abundant in Cells That Secrete Large Amounts of Protein Pancreatic Cell – secretes hormones

Smooth ER Functions Synthesis of Lipids for Membranes: Phospholipids and Cholesterol Storage site for Calcium - mediated by calcium binding proteins Synthesis of Steroid Hormones from Cholesterol in in Specialized cell types In Liver Cells, Detoxifies Wide Variety of Organic Compounds (alcohol, barbiturates )

The Smooth ER is Extensively Developed in Specialized Cell Types Leydig Cell of Human Testis (Steroid Hormone Secreting Cell) Liver Cells Skeletal Muscle Kidney Tubules and Steroid Producing Glands

Proteins Can Enter the Secretory Pathway by Translocation Across the ER Membrane 1.Co-Translational Targeting Requires Recognition of a Signal Sequence by Signal Recognition Particle (SRP) in the cytosol. Involves Translocation of Polypeptide through Gated Aqueous Channels called a Translocon Secretory proteins or lumen resident proteins – translocated completely across the ER membrane into lumen Transmembrane proteins are integrated into the ER membrane 2. Post-Translational Targeting

Proteins use Signal Sequences for Targeting to the ER Signal Sequence: For Soluble Proteins destined for ER Almost always N-terminal hydrophobic Approximately 20 primarily hydrophobic The ER Signal Sequence is Both Necessary and Sufficient to Target a Protein to the ER

Cytosolic Ribosomes are Directed to the ER Membrane for Translocation of Targeted Protein

Co-Translational Targeting of Protein to the ER Involves the Signal Recognition Particle (SRP)

Transmembrane Transport of A Soluble Protein into ER Lumen 2. Cleavage of Signal Sequence By Signal Peptidase Release of Protein Into Lumen 1. Translocon Binds Signal Sequence Transfer of Nascent Protein Across Membrane -Secretory Proteins -Lumen Resident Proteins

Integration of a Transmembrane Protein Into the ER Membrane Requires N-terminal ER signal sequence and a Stop Transfer Sequence

Formation of a Double Pass Membrane Protein Double Pass Membrane Protein- Internal ER signal sequence (Start Transfer Sequence) - and a Stop Transfer Sequence

Protein Folding and Processing in the ER Lumenal Resident Proteins are involved in the folding, assembly, and modification of newly translocated polypeptides -Protein Folding and Assembly into Multisubunit Proteins Hsp70 Chaperone Proteins- BiP Protein Disulfide Isomerase -Removal of the Signal Sequence -Attachment of a Complex Carbohydrate structure (N-linked Glycosylation) -Attachment of the phospholipid glycosylphospatidylinositol (GPI anchor)

The ER Synthesizes the Major Cellular Phospholipids Membrane Synthesis is Associated with pre-existing membranes Phospholipids on cytosolic side of ER; Glycolipids and Sphingomyelin on Lumenal surface of Golgi Scramblase- a phospholipid translocator- ensures symmetrical growth of bilayer Lumenal lipids become the outer leaflet of plasma membrane

Lipids and Proteins Can Exit the Transitional ER by Vesicular Transport

The Golgi Apparatus is a Series of Membrane Bound Compartments with Distinct Biochemical Functions Site of Carbohydrate Synthesis Processing, Sorting, and Dispatching of Various ER Products Lipid Synthesis Especially Prominent in Cells that Function In Secretion

Histochemical Stains Demonstrate the Biochemical Compartmentalization of the Golgi Apparatus Cis –osmium mannosidase II Trans-diphosphatase

Protein Glycosylation N-linked Glycosylation (typically N X S/T Sequence) Addition occurs in the RER upon transport- quality control checkpoint for proper protein folding Additional modifications (trimmings and additions) occur in Golgi O-linked – attached to OH group of Serine or Threonine Addition occurs in the Golgi

Protein Glycosylation Begins in the RER N-linked glycosylation: Addition onto an Asparagine Amino Acid

Protein Glycosylation and Sorting within the Golgi

Membrane-Bound Vesicles Transport Materials Through the Endomembrane System 1)Cargo Selection 2) Budding 3)Uncoating 4)Tethering 5) Docking 6) Fusion 7) Recycling

Membrane-Bound Vesicles Transport Materials Through the Endomembrane System Vesicles are enclosed by proteinaceous coats Identified coats: Clathrin, COPI and COPII Coat functions: 1)Helps membrane bud to form vesicle 2)Cargo Selection: Captures specific components for onward transport

Utilization of Different Coats in Vesicular Transport

Clathrin Coated Vesicle Formation Clathrin binds to the membrane Begins to assemble into a basket Causes curvature of the membrane (called a clathrin-coated pit) More clathrin binds and membrane continues to curve Eventually membrane “circle” closes Coated vesicle pinches off

Clathrin Triskelions Can Assemble into Closed Cages Network of hexagons and pentagons of triskelions Scaffold responsible for changes in shape of the coat

Vesicle Budding is Driven by Formation of Protein Coat Clathrin- No role in Cargo selection Adaptins- Secure Clathrin Coat Select cargo by capturing Cargo receptors GTP hydrolysis by Dynamin required For “Scission” – pinching off of vesicles

Scission Requires GTP Hydrolysis by Dynamin

Snares Help Direct Transport Vesicles to their Target Membranes For Fusion Vesicles that bud carry specific marker proteins called v-SNAREs Bind complementary t-SNARE on target membrane 20 Different SNARES ( complementary sets) in Animal Cells Each associated with particular membrane bound organelle

SNARE Pairing is Essential for Membrane Fusion Trans SNARE Complex Separation of SNAREs By NSF Requires ATP SNARE-SNARE Pairing provides the energy to bring two bilayers Sufficiently Close to destabilize them and result in fusion

About 60 different Rabs exist- each with Characteristic Distribution Inactive Rab GDP bound Cytosolic Form Active Rab: GTP bound Membrane Bound Rab Effectors- Concentrate and Tether Vesicle Near Target Site Rab Contributes to Accuracy of Vesicle Targeting Before Membrane Fusion is Allowed to Occur

Vesicle Trafficking is Both Anterograde and Retrograde Why do you need Retrograde Trafficking?

ER Retrieval Signals are Required to Retrieve Resident ER Proteins that Escape Returned via Retrograde Transport ER Membrane Proteins: KKXX Sequence- recognized by COPI Coated Transport Vesicles ER Soluble Proteins KDEL Sequence (Lys Asp Glu Leu) Recognized by KDEL Receptor ( which has a KKXX Sequence) COPI Coated Vesicle Transport Signals located at C-terminus of proteins

Models for Transport Through Golgi Apparatus Golgi Cisternae Stay in Place Anterograde transport of Cargo by Vesicles Cargo Stays within a Single Golgi Cisterna, which is Remodeled And Changes Position Probably a Combination of Both- May depend upon Cargo Size !!

The Exocytic Pathway: Destination Plasma Membrane or Extracellular Space Sorting Occurs in Trans Golgi Specific Signal Not Known for Targeting to Secretory Pathway Small molecules, mucus, Digestive enzymes, hormones Aggregation of molecules To be packaged –special Surface conditions- low pH Or high Ca2+ influences Plasma Membrane Proteins and Lipids

Lysosomes Function as the the Principal Sites of Intracellular Digestion Digests material primarily taken up by Endocytosis Single Membrane Organelle Has ATPase Driven H+ Pump to Maintain Acidic Lumen Lumen Contains Many Acid Hydrolases Lysosomal Membrane Proteins are highly Glycosylated -protects from Hydrolases in lumen EM Stained for Acid Phosphatase

Proteins synthesized in ER N-linked Glycosylated Transported to Golgi In Cis Golgi Network Recognition of Signal Patch in Protein Results in Addition of Multiple Mannose 6- phosphate Groups to N-glycosylation sites Trans Golgi- Recognition of M6P by Mannose-6-Phosphate Receptor Receptor binds Adaptins Assembly of Clathrin Coated Vesicles Transport to Late Endosome (M6P receptor is recycled back to Trans Golgi) To Lysosome Lysosomal Membrane Proteins Transport is M6P Receptor Independent Vesicular Transport of Soluble Lysosomal Resident Proteins

Endocytosis: Transport Into the Cell From the Plasma Membrane to Lysosomes Internalize Nutrients Regulate Cell Surface Expression of Proteins Uptake and Digestion of Cellular Debris Recovery of Membrane Inserted during Secretion Type Based on Size of Endocytic Vesicles Formed Pinocytosis- Ingestion of Fluid and Molecules via Small Vesicles (<200nm in diameter) Phagocytosis- ingestion of large particles (> 200nm in diameter)

Extracellular Molecules Taken up by Endocytosis are transported to Endosomes, which mature into Lysosomes

Cholesterol is Taken Up by Animal Cells by Receptor Mediated Endocytosis Cells outside of the liver and intestine obtain their Cholesterol from plasma rather than synthesizing de novo

Receptor Uptake Involves Transcytosis in Polarized Epithelial Cells

Enjoy your Spring Break!!