Cytoplasmic Membrane Systems IILecture 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 ReticulumGolgi Apparatus LysosomesEndosomes Vesicles

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 AttachedSynthesis 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

Approximately 20 primarily hydrophobic 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 NascentProtein 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- BiPProtein 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 isAssociated with pre-existingmembranes

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 Selection2) Budding3)Uncoating4)Tethering5) Docking6) 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 CoatSelect cargo by capturingCargo receptors

GTP hydrolysis byDynamin 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 complementaryt-SNARE on target membrane

20 Different SNARES ( complementary sets) in Animal CellsEach associated with particular membrane bound organelle

SNARE Pairing is Essential for Membrane Fusion

Trans SNARE

Complex

Separation of SNAREs

By NSFRequires

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 RabGDP boundCytosolic

Form

Active Rab:GTP bound Membrane

Bound

Rab Effectors- Concentrate andTether VesicleNear 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 ProteinsKDEL 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 PlaceAnterograde transport of Cargoby Vesicles

Cargo Stays within a Single GolgiCisterna, which is RemodeledAnd Changes Position

Probably a Combination of Both- May depend uponCargo 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, hormonesAggregation of molecules To be packaged –specialSurface conditions- low pH Or high Ca2+ influences

Plasma MembraneProteins 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

EMStained 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 NutrientsRegulate Cell Surface Expression of ProteinsUptake and Digestion of Cellular DebrisRecovery of Membrane Inserted during Secretion

Type Based on Size of Endocytic Vesicles Formed

Pinocytosis- Ingestion of Fluid andMolecules 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 intestineobtain their Cholesterol from plasma rather than synthesizing de novo

Receptor Uptake Involves Transcytosis in

Polarized Epithelial Cells

Enjoy your Spring Break!!