Cargo Packaging & Vesicular Fusion/Lysosomes

ceramides

Glycolipids and sphingomyelin are synthesized from ______________ in the Golgi.

Ceramide

This is a waxy lipid composed of a sphingosine and a fatty acid tail.

carbohydrates

The addition of ____________________ to ceramide yields a variety of different glycolipids.

phosphorylcholine; phosphatidylcholine

Sphingomyelin is synthesized by the transfer of a ________________________ group from ________________________________ to ceramide.

Trans-Golgi Network

In this network, molecules are packaged and sorted into transport vesicles.

membrane

Proteins that need to stay in the Golgi are associated with the __________________ & have signals that stop packaging/transport.

1. Direct transport to the plasma membrane

2. Recycling endosomes

3. Regulated secretory pathways

Transport from the Golgi to the cell surface can occur by 3 routes:

Recycling Endosomes

These facilitate movement from the Golgi back to the cell surface by acting as a sorting station, receiving vesicles from the trans-Golgi network & then selectively delivering specific cargo to the cell surface through a network of tubular membranes (recycles membrane components back to the membrane).

apical; basolateral

In polarized cells of epithelial tissue, plasma membranes are divided into ___________ domains and ______________ domains, each with specific proteins

Apical Domain

This domain is exposed to the lumen and is involved in nutrient absorption.

Basolateral Domain

This domain is in contact with neighboring cells and the extracellular matrix, and is involved in transferring nutrients into circulation.

peptide hormones; neurotransmitters

Regulated Secretory Pathways secrete ___________________________ and ___________________________.

Amylin

This is an example of a peptide hormone that prevents the release of sugar with insulin.

1. Small molecules- no connection to Golgi (glutamine, adrenaline, etc.)

2. Neuropeptides- get moved to axon

These are the types of hormones and neurotransmitters that regulated secretory pathways can secrete:

Apical; Basolateral

Proteins leaving the Trans-Golgi are packaged into ______________ or ___________________ Domains.

selectivity

The _____________________ of vesicular transport is key to maintaining the functional organization of the cell.

1. Isolation of yeast mutants defective in protein transport and sorting

2. Reconstitution of vesicular transport in cell-free systems

3. Biochemical analysis of synaptic vesicles

These are the 3 experimental approaches that have been used to understand the mechanisms that control vesicular transport:

• They are easy to track and manipulate

• Mutant yeast strains with altered protein domains give insights into functional roles of different protein regions in vesicular transport.

Why are yeasts good to study to understand vesicular transport?

It is a system that is fully controlled, and you can isolate transport proteins….you can also provide a functional analysis of the proteins identified by mutations in yeast.

Why are reconstituted (in-vitro) systems helpful for understanding vesicular transport?

Synapse

This is the junction of a neuron with another cell.

It allows researchers to identify and characterize the specific proteins present on the vesicle membrane.

Why is a biochemical analysis of synaptic vesicles helpful to understand the mechanisms that control vesicular transport?

secretion

Synaptic transmission in neurons is a specialized form of regulated _________________.

synaptic vesicles

Chemical Neurotransmitters are stored in the neuron in _________________________.

synaptic vesicles; synapse

Stimulation of the neuron triggers fusion of ________________________with the plasma membrane and neuro-transmitters are released into the ______________.

brain; isolated

Synaptic vesicles from __________ tissue can be purified in large amounts and proteins _____________.

1. Cells are tagged with GFP

2. DNA is transcribed → mRNA

3. proteins are produced and targeted

4. GFP proteins allow vesicles carrying proteins to be seen in the vesicles

Explain the process of Immunostaining to view vesicular transport:

cytosolic coat

Transport vesicles from the ER are coated with ______________________________ proteins.

cytosol

Cytosolic coats assemble as the vesicle buds off and are removed in the _____________ before it reaches its target.

1. COPII-Coated Vesicles

2. COPI-Coated Vesicles

3. Clathrin-Coated Vesicles

These are the 3 families of vesicle coat proteins:

COPII-Coated Vesicles

These carry proteins from the ER to the ER-Golgi intermediate complex & onto the Golgi.

Protein Complex II.

COPII-Coated Vesicles are coated by Coat __________________________.

Anterograde Transprot

COPII-Coated Vesicle transport is called ___________________________.

COPI-Coated Vesicles

These bud from the ER-Golgi intermediate compartment or Golgi & carry their cargo back, returning proteins to their earlier compartments.

Protein Complex I

COPI-Coated Vesicles are coated by Coat _______________________.

Retrograde Transport

COPI-Coated Vesicle Transport is called ______________________________________.

Clathrin-Coated Vesicles

This kind of vesicle transport is in both directions between the Trans-Golgi Network, Endosomes, Lysosomes, and the Plasma Membrane.

GTP-binding

The formation of coated vesicles is regulated by small __________________ proteins (ARF1 and Sar1).

adaptor; cargo

GTP-binding proteins recruit ______________ proteins that mediate vesicle assembly by interacting with the __________ & coat proteins.

Clathrin

Interactions between adaptor proteins and ____________ are responsible for Clathring vesicle self-assembly (Clathrin itself doesn’t interact with cargo).

C-termini

Heavy Clathrin polypeptide chains are associated by their ______________ forming a triskelion.

polymerization

Light Clathrin polypeptide chains modulate heavy-chain _________________________.

2

The human Clathrin has ______ polypeptide chains.

Triskelia

This is flexible & assembles into hexagons & pentagons; no two structures are the same (different vesicle sizes).

1. The vesicle must recognize the correct target membrane

2. Vesicle and target membrane must fuse, delivering the contents to the target organelle

2 thing must happen for fusion of a transport vesicle with its target:

Rab Proteins

These are small GTP-binding proteins that direct vesicles to the correct membrane.

coat; surface

Rab Proteins interact with the vesicle _______ and reside on the cytoplasmic ____________ of the vesicle.

tethering

Fusion between transport vesicles and target membranes is mediated by __________________ factors and Rab proteins.

SNAREs

These are proteins that mediate vesicle fusion.

v-SNARE

This kind of SNARE is embedded in the vesicle membrane.

t-SNARE

This kind of SNARE is embedded in the target membrane.

fusion

A complex between both SNARES is required for _________________.

t-SNARES

__________________ guide v-SNARES during the complex formation.

alpha-helical; 4

SNARE proteins have a central _______________ domain. This domain binds to other domains with ________ SNARE molecules form a ________ membered supercoil.

Domain

____________ interactions zip SNAREs on vesicle and target membranes together.

pairing

SNARE-SNARE _____________ provides the energy to bring the two bilayers close enough to destabilize them & fuse.

Lysosome

This is a membrane-bound organelle that contains digestive enzymes (break down worn-out parts, destroy invaders).

Lysosomes

Yeasts, Fungi, & Plants lack ________________.

Vacuole

This has the same function as the lysosome, plus nutrient storage and maintaining turgor pressure.

Lysosomal Storage Diseases

This occurs when undegraded material accumulates in the lysosomes of affected individuals.

Gaucher Disease

This is a disease caused by a deficiency in Glucocerebrosidase, which catalyzes the hydrolysis of glucoceramide to glucose and ceramide.

macrophages

In the most common forms of Gaucher, only the __________________ are affected, their job is to eliminate aged & damaged cells.

acid hydrolases

Most lysosomal enzymes are __________________—active at
pH 5 in lysosomes, preventing uncontrolled digestion of cell contents if lysosome membrane breaks down.

proton pump

To maintain the acidic pH, a __________________ in the
lysosomal membrane actively transports protons into the
lysosome.

endosome

Lysosomes are formed when transport vesicles from the trans-Golgi network fuse with a late ______________.

1. Early Endosomes

2. Recycling Endosomes

3. Late Endosomes

These are the 3 types of Endosomes:

1. Early endosomes fuse with endocytic vesicles from plasma membrane.

2. They separate molecules for recycling from molecules for degradation in lysosomes.

3. Molecules to be recycled are passed to recycling endosomes & back to the plasma membrane.

4. Molecules destined for degradation are transported to multivesicular bodies, then to late endosomes.

5. Transport vesicles carrying acid hydrolases from the trans-Golgi network, then fuse with late endosomes, which mature into lysosomes.

These are the steps of Lysosome formation:

Phagocytosis

This is when specialized cells, such as macrophages, take up & degrade large particles.

Autophagy

This is the turnover of the cell’s own components; important in embryonic development.

autophagosome

In autophagy, a small area of the cytoplasm/organelle is enclosed in a vesicle called the ___________________, which fuses with a lysosome, & its contents are digested.

phagosome; phagolysosomes

In phagocytosis, particles are taken up in phagocytic vacuoles called the __________, which fuse with lysosomes to become _____________________.