5. Lysosome, Endocytosis and Secretion

some modifications are made to carbohydrates in the ___

trans-golgi network

Trans-Golgi Network (TGN) sorting pathways

1. signal mediated diversion to lysosomes

2. constitutive secretory pathway

3. signal mediated diversion to secretory vesicles (for regulated secretion)

__ is the sorting compartment of the Golgi

• controls __

Trans-Golgi NEtwork (TGN)

• the flow of vesicles outward toward the plasma membrane

(TGN): if not added signal, proteins move on to __

• this dumps lumen contents __

• puts membrane proteins in __

secretory vesicles which fused with the plasma membrane (constitutive secretion/exocytosis)

• outside the cell

• the plasma membrane

(TGN): if a signal is present, the protein is ___

diverted

• lysosomes

• regulated secretion/exocytosis

lysosomes are ___ that take part in ___

cytoplasmic vesicles, degradation of macromolecules

describe internal environment of lysosomes

acidic due to a membrane pump that transports H+ in using ATP as an energy source

contents of the lysosome are ___ that work in acidic environment (__)

• __ - degrades protein

• __ - degrades DNA

• __ - degrades RNA

• __ - degrades polysaccharides

hydrolytic enzymes (degrade macromolecules)

• Protease

• DNase

• RNase

• Glycosidase

the hydrolytic enzymes of lysosomes are delivered from __

trans golgi network

the material degraded by lysosomes can be transported to the cytoplasm and used for ___

synthesis or dumped out of cell

Lysosome Formation

• vesicles containing lysosomal components from the ___ fuse with ___

• these then fuse with existing lysosomes, creating __ and allowing the degradation of ___

• these then mature again into ___, which will await another delivery from LEs

• TGN; late endosomes (LEs)

• endolysosomes; endocytosed material

• classical lysosomes

how are lysosomal proteins targeted to the LEs/Lysosomes?

a “mannose-6-phosphate” signal

PROCESS OF LYSOSOMAL PROTEINS BEING TARGETED TO LEs/LYSOSOMES

• a __ in the lysosomal protein is recognized by an enzyme in the cis-golgi

• a __ adds a modified phosphate mannose on the protein’s oligosaccharide tree

• creates a ___ signal

• this signal is recognized by a ___

• __ bind to the cytoplasmic domain of this receptor, thus sorting the proteins into __

• Rabs…SNARES…fusion!

• in the lysosome, the pH ___

• in the lysosome __ is removed so the targeting signal is no longer there

• the receptor recycles back to the __

• signal patch

• phosphotransferase

• mannose-6-phosphate signal

• transmembrane Man-6-P receptor in the Trans Golgi Network

• Clathrin coat and adapter proteins; vesicles destined for the late endosome/lysosome path

• drops, causing receptor to release the protein

• the phosphate

• TGN!

what does a phosphotransferase do

its an enzyme that can transfer a phosphate group onto a target

the first coat protein discovered

Clathrin

what is clathrin made of

3 heavy chains and 3 light chains that form Clathrin triskellions

cool ability that clathrin has

can self assemble in test tube (in vitro) into polyhedral cage

clathrin forms a ___ around a vesicle

very cool looking cage

rare human genetic diseases lead to formation of inclusion bodies in cells… what does this mean?

persistent vesicles full of stuff that is normally broken down by lysosomes

what is I-cell disease

specific, severe case in which none of the lysosomal enzymes are sorted correctly to lysosomes

the multiple signal and receptor recognition events in lysosomal targeting

• hydrophobic signal sequence/SRP & receptor targets protein to ER

• glycosylation site (Asn-X-Ser/Thr)

• ER export sequences for cargo receptors that sort lysosome proteins into COPII vesicles bound for CGN

• signal patch; phosphotransferase leads to Man-6-P addition in cis-golgi

• Man-6-P; Man-6-P receptors in the TGN or plasma membrane sort proteins into clathrin-coated vesicles bound for late endosomes/lysosomes

what is endocytosis

movement of materials into the cell by plasma membrane-derived vesicles

two types of endocytosis

1. pinocytosis (aka cell drinking)

2. phagocytosis

what is pinocytosis

uptake of fluids and small, soluble materials from outside the cell

what is phagocytosis

engulfment of larger solid material from outside by direct attachment

material from both pinocytosis and phagocytosis can end up in ___

lysosomes

PHAGOCYTOSIS

• cell surface “zippers” around the particle to engulf it

  • contact of particle, bacteria, etc with __ stimulates the uptake

  • recognition is through ___

  • an outward protrusion of ___ rather than the inward budding of pinocytosis

• vesicle is then closed and internalized to form a __

• fusion with lysosome introduces enzymes into ___ to digest contents

• used as a ___ mechanism for protists

• used as a ___ mechanism for mammals

• surface receptors

• surface molecules on the particles, immune antibodies, or complement proteins coating it

• membrane driven actin filaments

• phagosome

• phagolysosome

• defense

• defense or homeostasis

examples of how phagocytosis is used in mammals

1. immune cells (macrophages and neutrophils) eat and destroy bacterial invaders

2. used to get rid of foreign material in a wound

3. used to safely remove damaged, defective, or dying cells

   • normally cells know not to phagocytose each other

PINOCYTOSIS

• “cell drinking”, but __

• some is ___ and may be non-specific

  • brings in ___

  • balances the ___

• some is ___ and may be specific

  • i.e ___

• brings in soluble materials too

• constitutive (constant)

  • nutrients and soluble macromolecules

  • outward flow of membrane by secretion

• regulated

  • receptor-mediated endocytosis

two main modes of entry for pinocytosis

1. caveolae (small)

2. clathrin-coated pits (larger)

what is caveolae

small, lipid raft-based ivaginations of the plasma membrane stably coated by a protein called caveolin

with caveolae, there is no ___ so ___ is not well understood

obvious dynamic coat recruitment; budding

clathrin coated pits and receptor mediated endocytosis

RECEPTOR MEDIATED ENDOCYTOSIS

• clathrin coated-pit formation and uptake of ligands triggered by ___:

  • receptors associate with ___

  • some can associate without ligand, some only when bound

  • __ nucleate clathrin assembly

  • more receptors can continue to move in and __

  • clathrin assembly caused the pit to ___

  • non-specific membrane proteins that dont associate with coat proteins arent kept in pits and thus ___

• the binding of the ligands to transmembrane receptors on the cell surface

  • clathrin coat adaptor proteins (AP2)

  • receptors, adaptor proteins, PIP2

  • interact with coat proteins in newly forming pits

  • invaginate and ultimately the vesicle is released inside the cell

  • arent taken up very often

what is the early endosome

very important sorting compartment

some receptors are recycled during ___

receptor mediated endocytosis

LDL processing by cells

(Receptor Mediated Endocytosis)

• ___ move cholesterol from liver to other body cells

  • large protein + __

• ___ in coated pits bind LDL

• internalized by receptor-mediated endocytosis

• the early endosome is a sorting compartment

  • receptor is ___

  • the LDL continues on to ___

• Low Density Lipoproteins (LDL)

  • micelle of lipid and cholesterol

• LDL-Receptor

  • sorted and sent back to surface

  • lysosomes for processing

individuals with genetic disorders of cholesterol metabolism have defects in the ___

delivery machinery

• if you have a defective receptor or lack on, you dont take up cholesterol efficiently leading to a high blood and low cellular concentration (hypercholesterolemia)

what is Hypercholesterolemia

high blood and low cellular concentration

some receptors can be targeted for degradation: ___ can modulate signal transduction pathway (ie RTKs)

receptor downregulaton

membrane proteins destined for degradation are ___ and targeted to ___

marked by ubiquitination; topologically distinct, intraluminal vesicles in late endosomes

___ allow for targeted membrane proteins to be degraded while protecting other membrane proteins

• basically allows stuff deep inside to be destroyed but not the stuff around it

multivesicular bodies

how are multivesicular endosomes formed

ubiquitination and ESCRT

• ubiquitin group on membrane proteins allows ESCRT complex to gather up and shepherd membrane proteins into forming intraluminal vesicles

MVB

multivesicular bodies

what are exosomes

extracellular vesicles that can be released from cells by fusion of MVBs with the plasma membrane

PATHWAYS TO DEGRADATION IN LYSOSOMES

1. Endocytosis → early endosome → late endosome → lysosome

2. Phagocytosis → phagosome → lysosome

3. Autophagy → autophagosome → lysosome

Autophagy (self eating) delivers ___

cytosolic proteins or entire organelles to lysosomes by surrounding it in double membrane

what happens during exocytosis/secretion

vesicles bud from the TGN and move to the plasma membrane

different types of exocytosis/secretion

1. Constitutive secretion

2. Regulated secretion

Constitutive secretion: secretory vesicles move to the plasma membrane and fuse, ___

• relatively __ rate

dumping their contents outside the cell or contributing membrane proteins to the plasma membrane

• constant

Regulated secretion: the vesicles are held near the membrane until ___

• the secretory vesicles accumulate in the ___

• when triggered by an external signal, they ___

• in some cases, this is done to ___

  • ex: nerve impulse leads to secretion of neurotransmitter

• in some cases, this is done to ___

  • ex: insulin causes more glucose transporter to be put on the surface

a stimulus is received and then they fuse. the signals that sort proteins for regulated secretion are not well understood but they often lead to aggregates of vesicle contents in a low pH environment

• cytoplasm

• fuse with the plasma membrane

• release a soluble protein in the vesicle into the outside environment

• put a membrane protein onto the surface of the cell

vesicles sort to different plasma membrane domains using ___

• __ have been shown to act as apical signals

• __ can act as apical or basal signals, based on cell type

• some __ act as direct basal sorting signals

• distinct __ and __ are involved in vesicle targeting between domains

a variety of mechanisms in specialized cells

• N- and O- linked sugars

• Glycosphingolipids and GPI-linked proteins or lipid rafts

• amino acid sequences

• Rabs and SNARES

OVERVIEW 1: Signals in protein trafficking in the endomembrane system

• proteins are made on cytoplasmic ribosomes

• if a hydrophobic start transfer sequence is made, the protein enters the ER/Golgi

• Asn-X-Ser signals N-linked glycosylation

• most things move to the Golgi

• if a protein has an ER retention signal, it recycles back to the ER

• if it does not, it goes on in the Golgi

• material “moves” through the golgi apparatus by cisternal maturation

• retention in the golgi is not well understood

• if a protein has a lysosome signal patch it gets a Man-6-P and is sorted in the TGN to lysosomes

• otherwise, it continues on to exocytic, secretory vesicles

  • if it is a membrane protein, it ends up in the plasma membrane (LDL receptor)

  • if is in vesicle lumen, it is secreted

• sorting signals for regulated secretion still poorly understood

• apical and basal sorting signals help organize epithelial cells

OVERVIEW 2: Secretion, Endocytosis, and Membrane dynamics

• Outward flow by secretion

  • Membrane, membrane proteins, and soluble proteins in vesicles move from ER to Golgi to secretory vesicles to plasma membrane; Membrane and membrane proteins contribute to plasma membrane while soluble proteins are released from the cell

  • Some material is diverted to lysosomes by Man-6-P signal

  • Other material is delivered to secretory vesicles

    • Some vesicles travel to plasma membrane and fuse (constitutive)

    • Others move to plasma membrane but await a signal to fuse (regulated)

• Inward flow of material through endocytosis and phagocytosis

  • Endocytic vesicles package soluble materials and remove membrane and membrane proteins from the plasma membrane.

  • Some material fuses with lysosomes for degradation

  • Some material is returned to the plasma membrane

  • Inward flowing material can also be diverted to other compartments (transcytosis)

• Recycling and sorting of proteins and membrane can occur to return them to
  their correct functional locations.

• This is a very dynamic process. The two systems must balance otherwise
  you will increase the amount of plasma membrane and lose internal
  membrane or vice versa.