Cytoplasmic membrane part 2

Cell Biology

TGN

(trans-Golgi network) the Major Processing Center where Proteins Destined for Different Sites are Sorted into Different Membrane-Bound Carriers

The best understood post-Golgi pathway involves

Lysosomal enzymes

Where are Lysosomal enzymes synthesized?

they are Synthesized in the RER and Carried to the Golgi, Where they are Recognized Due to Signal Patches (Signals that are Part of their 3D structure)

How are lysosomal enzymes recognized?

by Enzymes that Catalyze the 2-Step Addition of a Phosphate Group to Certain Mannose Residues that Act as a Recognition Signal

Lysosomal Enzymes Carrying Mannose-6-Phosphate Signal are recognized by what?

they are Recognized and Captured by Mannose-6-Phosphate Receptors, that are Integral Membrane Proteins of the TGN

How are lysosomal enzymes transported from the TGN?

in Clathrin-coated vesicles who has a Honeycomb-Like Lattice of the Protein Clathrin

What does clathrin do for adaptor proteins?

Forms a Scaffold for an Inner Shell of Adaptor Proteins that Cover the Surface of the Vesicle that Faces the Cytosol

What proteins escort lysosomal enzymes from the TGN

adaptor proteins like GGAs or AP complexes

AP-1 and GGAs

are Associated with TGN Vesicles Moving to Early, Late or Recycling Endosomes

AP-2

is Involved with the Endocytosis of Vesicles from the Plasma Membrane to Early Endosomes

AP-3

is Involved with the Movement of Vesicles from Recycling (Tubular) Endosomes to Late Endosomes

AP-4

is Involved with the Movement of Vesicles Between the TGN, Early, Late and Recycling Endosomes

AP-5

is Involved with the Movement of Vesicles Between Late Endosomes and Lysosomes

retromer coat complexes

are Involved in Vesicles Returning Mannose-6-Phosphate Receptors from Early or Recycling Endosomes to the TGN

TIP47 (perilipin)

is Involved in Vesicles Returning Mannose-6-Phosphate Receptors from Late Endosomes to the TGN

Which AP complexes can associate with Clathrin?

The AP complexes that can associate with Clathrin include AP-1, AP-2, and AP-3, AP-4

Clathrin-Coated Vesicles have Adaptor Proteins Like GGA, that are Recruited to the Membrane by

ARF1-GTP (GGA can then recruit AP1)

Adapter proteins bind to

the sorting signal in the membrane proteins such as the mannose-6-phosphate receptor, helping to recruit clathrin and other coat proteins for vesicle formation.

what are mannose-6-phosphate receptors bound to?

soluble lysosomal enzymes in the vesicle lumen

the G-protein ARF1 Effectively Allows

the Binding of Coat Proteins at the Site where ARF1 is Itself

Bound to the Membrane

Once the Vesicle Buds from the TGN

ARF1’s GTP is Hydrolyzed to GDP, Releasing the Coat, the Clathrin is Shed into the Cytoplasm Again The Uncoated Vesicle then Fuses with Early, Late or Recycling Endosomes

What happens in endosomes with lower pH, particularly late endosomes?

the Mannose-6-Phosphate Receptors Release the Bound Soluble Lysosomal Enzymes who Carry on their Route to the Lysosome, or the Late Endosome Fuses with a Lysosome; The Mannose-6 Phosphate Receptors are Returned to the TGN in Retromer or TIP47 Vesicles

What can mannose-6-phosphate receptors do for the plasma membrane?

they can be sent there to retrieve certain secreted enzymes from outside the cell using endocytosis

Secretory and Membrane Proteins Destined for the Plasma Membrane Also Leave the TGN in

vesicles or secretory granules

why do secretory or membrane proteins destined for the plasma membrane go through the TGN

Here the Proteins Concerned are Thought to Form Aggregates which are then Partitioned into Particular Membrane Bodies as the TGN Fragments into Vesicles/Secretory Granules

The Delivery of Integral Proteins to the Plasma Membrane Occurs Due to

Sorting Signals Present in their Cytoplasmic Tails

The Sorting Signals for Each Membrane Domain are Different and Sorting Occurs in the

TGN or recycled endosomes

What makes basolateral proteins targetable?

Short Hydrophobic Amino Acid Sequences Such as Di-Leucine (LL) or Tyrosine Containing Motifs

what makes apical proteins targetable?

although not fully understood but involves carbohydrate modifications rather than amino acid sequences

a known apical membrane protein sorting mechanism

Transcytosis, Where Proteins are Delivered Initially to the Basolateral Membrane Before Being Endocytosed

endocytosed proteins are re-sorted via

Early Endosomes and Sent to the Apical Membrane

Plasma Membrane Proteins in Non-Polarized Cells Do Not Need

Any Kind of Special Sorting Machinery and Can Just be Carried to the Surface in Vesicles of the Constitutive Secretory Pathway

selective fusion

One Factor that Ensures a Directed Flow Through the Various Endomembrane Compartments Starting at the ER

parts of the vesicle fusion process

1) Movement of Vesicles Towards the Target Compartment For Fusion is Mediated by Microtubules

2) Vesicles are Often Tethered Once they Reach the Target Compartment by Extended Fibrous Proteins

3) At Some Point During Fusion the Membranes Come Close to Each Other

4) When Artificial Liposomes Containing v- or t-SNAREs are Mixed, the 2 Types of Vesicles Fuse Together, but Not Vesicles of the Same Kind

compatibility between the vesicle and the target compartment is determined by members of the

Rab G-Protein Family

Rabs associate with membranes by a

lipid anchor

When GTP is Bound to the Rab G-Proteins, they are Thought to

Recruit Rab-Effector Tethering Proteins to the Membrane Surfaces

Griscelli Syndrome

Patients Exhibit Partial Albinism and are Immuno-deficient, is Caused by Mutations in Rab27a, Rab27a Plays a Role in Transport of Pigment Containing Vesicles in Skin and Hair and in Exocytosis in T Lymphocytes

At Some Point During Fusion the Membranes Come Close to Each Other due to

Integral Proteins Called SNAREs in the 2 Membranes

SNAREs Vary in Structure and Size, But All Contain a Segment in their Cytosolic Domain Called

a SNARE Motif

v-SNAREs

Become Incorporated into the Vesicle During Budding

t-SNAREs

Located in the Target Membrane Compartment

The Best Studied SNAREs are

those Involved in Docking Synaptic Vesicles During Nerve Cell Neurotransmitter Release

The Plasma Membrane of the Nerve Cell Contains 2 t-SNAREs, Called

Syntaxin and SNAP-25

the Synaptic Vesicle Membranes Contain a Single v-SNARE

a Member of the Vesicle Membrane Associated Protein (VAMP) Family, Called Synaptobrevin

the Membranes Approach One Another, the SNARE Motifs of Opposing v- and t-SNAREs form a

4-Stranded Bundle, Each Bundle Consists of 4-Alpha Helices, 2 from Snap-25 and 1 Each from Syntaxin and Synaptobrevin, Together these Helices Form the Coiled Coil that Pulls the 2 Membranes into Close Proximity

The 4 SNAREs in the Bundle in Nerve Cells are Locked in an Inactive Conformation by Another Bound Protein Called

Complexin

Vesicles with SNAREs Inactivated by Complexin, are

Docked at the Membrane Ready to Discharge their Contents Virtually Instantly

The Activating Signal which Causes Vesicle Fusion

is a Rise in Intracellular Ca2+ Ion Concentration

Nerve Cells, Vesicle Fusion is Regulated by the Calcium Binding Protein,

Synaptotagmin Present in the Synaptic Vesicle Membrane, After a Rise in Intracellular Calcium, Synaptotagmin Displaces Complexin, Which Releases the Inhibition of the SNARE Complex, Causing Vesicle Fusion and Neurotransmitter Release

Dissociation of the 4-Stranded SNARE Bundles is Achieved by a

Doughnut-Shaped Cytosolic Protein Complex, Containing NSF and α-SNAP Proteins, these Attach to the Bundle and Twist it Apart Using Energy From ATP

exocytosis

Fusion of a Secretory Vesicle or Granule with the Plasma Membrane

exocysts

Protein complexes that mediate exocytosis by guiding vesicles to the plasma membrane for fusion.

exocysts consist of

8 Proteins (Sec3, 5, 6, 8, 10, 15 and Exo70 and 84) Plus a Rab Protein in the Vesicle, Sec3 and Exo 70 are Located on the Plasma Membrane, the Rest are on the Docking Vesicle

a typical lysosome contains

50 Hydrolytic Enzymes Produced in the ER and Shipped to Lysosomes in Vesicles

Lysosomal Enzymes All Have an Optimal Activity at an Acid pH and are thus

Acid Hydrolases

The Internal Lysosomal pH is Maintained by

a Membrane-Bound V-Type, H+-ATPase Pump

Mutations in the Genes that Encode Lysosomal Enzymes are Responsible for 40 Different Genetic Diseases Called

Lysosomal Storage Diseases that Affect 1 in 5000 Births,

Lysosomal Storage Diseases

These Lead to the Accumulation of Non-Degraded Products that Leads to a Build up in the Size and Number of Lysosomes

Gaucher’s Disease

a Mutation in the Gene for an Enzyme Required for the Breakdown of Glycolipids

phagolysosomes

are formed by the fusion of phagosomes containing engulfed material with lysosomes, allowing for the degradation of the contents,

envelopment of pseudopodia surrounding microbes involves

Phosphorylation of Membrane Phospholipids PI(4,5)P2 to Form PI(3,4,5)P3

Once Engulfed, Microbes May be Killed by

Lysosomal Enzymes or Oxygen Free Radicals Generated in the Lumen of the Phagosome

Engulfment of Materials Involves the Activities of

Actin Microfilaments Underneath the Plasma Membrane

Mycobacterium tuberculosis is Taken into Macrophages by Phagocytosis,

but the Phagosomes Fail to Fuse with a Lysosome, The Bacteria Inhibits Membrane Fusion and Instead Multiplies Inside the Cell

Q Fever, Coxiella burnetii, Becomes Enclosed in the Phagosome,

but Neither the Acidic Environment or the Lysosomal Enzymes Can Destroy it

Listeria monocytogenes, a Bacterium that Causes Meningitis,

Produces Proteins that Destroys the Lysosomal Membrane, This Allows the Listeria Bacterium to Escape into the Cell Cytoplasm

residual body or lipofusin pigment granule

phagolysosomes that contain undigested material leftover from phagocytosis. These structures can accumulate in cells and indicate previous immune responses.

autophagy

is a cellular process that degrades and recycles cellular components through the formation of autophagosomes that fuse with lysosomes.

Autophagic Vacuole

A double-membrane structure in cells formed during autophagy that encloses cellular components targeted for degradation and recycling.

autophagolysosome

organelles like mitochondria are initially surrounded by the double membrane part of the ER forming the autophagic vacuole, the ER then fuses with a lysosome to form this

Once the Digestive Process of the Autophagolysosome is Completed,

the Organelle Becomes a Residual Body

tonoplast

a membrane-bound structure in plant cells that encloses the central vacuole. It regulates the movement of ions and molecules in and out of the vacuole and helps maintain turgor pressure.

Vacuoles have Homologues of

Lysosomal Acid Hydrolase Enzymes

in vacuoles, internal pH is kept low by

Tonoplast V-Type H+-ATPases Pumping Protons into the Vacuole

Where are vacuole proteins made?

Vacuole proteins are synthesized in the endoplasmic reticulum (ER) and processed in the Golgi apparatus before being transported to the vacuole.

Proteins are Targeted to Vacuoles By

Short Peptide Signals Rather Than Carbohydrates (Mannose-6-Phosphate) as in Lysosomes

endocytosis

is where the Cell Internalizes Membrane Receptors and Their Bound Ligands

phagocytosis

Involves the Uptake of Particles from Extracellular Fluid

the endocytic pathway starts with

the Invagination of the Plasma Membrane to Form Cytoplasmic Vesicles that are Transported into the Cell

Bulk-Phase Endocytosis or Pinocytosis

is the Non-Specific Uptake of Extracellular Fluid, any molecules in the enclosed fluid enter the cell

Pinocytosis Also Removes Plasma Membrane and Functions Primarily to

Recycle Membrane Between the Cell Surface and the Interior

Receptor-Mediated Endocytosis (RME)

Absorbs Specific Macromolecules (Ligands) into the Cell, as they are Associated with Receptors on the Cell Surface

Clathrin-Coated Pits

are specialized structures in the plasma membrane that facilitate receptor-mediated endocytosis by assisting in the internalization of specific ligands.

Each Clathrin Molecule Consists of 3 Heavy Chains and 3 Light Chains Joined Together to Form a

triskelion

The Clathrin Triskelion is a Very Adaptable Structure for

Forming Polyhedral Shapes, as the clathrin curves it restructures and some of the hexagons become pentagonal

Like Clathrin Coated Vesicles Budding From the TGN, Endocytic Vesicles Also have

AP-2 Adaptor Complexes Between the Clathrin and the Vesicle Membrane

AP-2 Adaptors are a Complex of Multiple Subunits,

Consisting of α, β, σ, μ

Membrane Phosphotidyl-inositide PI(4,5)P2

recruits AP2 to the membrane and Also Changes AP2’s Conformation to Activate (Open) it so that the μ Chain Engages the Cytoplasmic Tails of Specific Plasma Membrane Receptors Leading to their Concentration (and their Bound Ligands) in the Vesicle, the β Adaptin Subunit Binds and Recruits the Clathrin Molecules Overlying the Adaptors

how many different kinds of PI are ther?

There are 7 Different Kinds of PI, [PI(3)P, PI(4)P, PI(5)P,

PI(3,4)P2, PI(4,5)P2, PI(3,5)P2 and PI(3,4,5)P3] Which have Phosphates Attached at Different Positions

In Endocytosis, AP2 (and Dynamin) Recruitment to the Membrane is Facilitated by

PI(4,5)P2

PI(3)P is

Localized to Early Endosomes and Late Endosome Intraluminal Vesicle Membranes

PI(4)P is

localized to the TGN, Secretory Granules, and Synaptic Vesicles

PI(3,5)P2 is

Localized to Late Endosome Boundary Membranes

PI(3,4,5)P3 is

Involved in the Later Stages of Phagocytosis

dynamin

is a G-Protein Required for the Release of Clathrin-Coated Vesicles from the Plasma Membrane

Dynamin Self-Assembles into

a Helical Collar Around the Neck of the Invaginated Coated Pit, Just Before it Pinches Off From the Membrane

Hydrolysis of GTP Bound to Dynamin

Induces a Conformational Change that Severs the Vesicle from the Membrane either directly or through the action of another protein

If a Non-Hydrolyzable Analogue of GTP (GTPγS) is Bound to Dynamin,

It Continues to Build the Helical Ring of Dynamin Subunits Without Severing Off the Vesicle

the two types of receptors that utilize the endocytic pathway

Housekeeping Receptors that Bring Nutrients into the Cell and Signaling Receptors