Endocytic pathways

Secretory pathways

Pathways of vesicular transport in the cell

Import

Export

Retrieval pathways

Recycling

ER Golgi PM

The secretory pathway delivers to the cell surface:

•Membrane (lipids)•Membrane proteins•Soluble proteins ( Secreted proteins )

exocytosis of GFP-linked membrane protein

Transport vesicles Golgi PM

default

ER Golgi PM

ER

• Synthesis of proteins (rER)

• Disulfid bridges

• “Standard-” glycosylation of proteins

Golgi

• Modification of sugars

• Sorting of proteins

Overview secretory pathway

Many proteins are glycosylated in the ER by addition of a preformed ‘sugar tree’

The oligisacharide is transfered to the N of the asparagine side chain recognition signal: Asn-X-Ser, Asn-X-Thr

N-linked glycosylation (most common type of glycosylation)

Oligosacharides are modified in the Golgi apparatus

ER

Golgi

ER Golgi PM

ER

• Synthesis of proteins (rER)

• Disulfid bridges

• “Standard-” Glycosylation of proteins

Golgi

• Modification of sugars

• Sorting of proteins

Overview secretory pathway

Exit of ER: Quality control

Incorrectly folded proteins, are incompletely assembled protein complexes are retained in the ER…

..and finally destructed in the cytoplasm

ERAD

ER-associated destruction

The Golgi apparatus is composed of several closed compartments

Cis Golgi networkCis cisterna

trans cisternaTrans Golgi network

cisternacisterna

cisterna

acidic

weakly acidic

A Golgi stackContains 3-20 cisternae.

A cell might have 1 large - 100 small stacks.

Two models for the transport of proteins from one cisterna to the next

• Cisternae are static organelles

• Contain resident proteins

• Secreted proteins are transport by vesicles between cisternae

• Cisternae are dynamic organelles moving through the stacks (maturing)

• resident Golgi proteins are recycled by vesicular transport

Cisternal maturation model Vesicular transport model

early cisterna

late cisterna

Yeast cell expressing fluorescence-labeled proteins, resident in early and late cisternae of the Golgi.

Golgi stack

Imagine that you

• labeled early cisternae green and late cisternae red by expression of fluorescence-tagged Golgi-residend proteins

• could watch individual cisternae in a living cell over a period oftime

What do you expect to see ifa) the vesicular transport model was trueb) the cisternal maturation model was true

early cisterna

late cisterna

Yeast cell expressing fluorescence-labeled proteins, resident in early and late cisternae of the Golgi.

Golgi stack

Imagine that you

• labeled early cisternae green and late cisternae red by expression offluorescence-tagged Golgi-residend proteins

• could watch individual cisternae in a living cell over a period of time

What do you expect to see ifa) the vesicular transport model was trueb) the cisternal maturation model was true

early cisterna

late cisterna

Yeast cell expressing fluorescence-labeled proteins, resident in early and late cisternae of the Golgi.

Golgi stack

Golgi maturation visualized in living yeast (Nature, 2006)

Movie shows two individual cisternae

The different functions of glycosylation

In ER:Retention of proteins in ER until they are properly folded

At cell surface: glycocalix

•protection from mechanical and chemical damage

•lubrication (slimy surface)

•cell-cell recognition, cell adhesion

Recognition of cell-surface carbohydrates on neutrophils is the first stage of their migration out of the blood stream at sites of infection

Functions of glycosylation:

Lumen of membrane-bounded compartments corresponds to the exterior

The constitutive and regulated secretory pathway

Constitutive secretory pathway• “Default” pathway, no signals required• Operates continuously in all eukaryotic cells

constant delivery of proteins and membrane lipids to the cell surface

The constitutive and regulated secretory pathway

Regulated secretory pathway• Signal dependent (proteins are recognized in the TGN)• Operates in specialized cells

Allows fast release of a large amount of proteins upon an extracellular signal

Proteins that aggregate under TGN conditions (acidic, high Ca2+)are recognized and sorted into secretory vesicles.

Formation of secretory vesicles

• In insulin-secreting β-cells from the pancreas, insulin is stored in highly concentrated form in secretoryvesicles (large dense core vesicles, diameter about 300 nm)

• Proteins and prohormones (e.g. neuropeptides) are often proteolytically processed during formation of secretory vesicles

Black dots: immunogold-labled clathrin molecules

Mast cell containing vesicles storing histamine before and after stimulation

Other cells that are specialized in the regulated exocytosis:Pancreatic cells secreting insulin

Special case: synaptic vesicles of neurons

before after

Protein sorting in the trans-Golgi network (TGN)

The different functions of glycosylation

In Golgi:

• Sorting signals for transport vesiclese.g. Mannose-6-phosphat

Late endosomes->lysosome

Mannose-6-phosphate (M6P) is a lysosomal sorting signal

What happens to acid hydrolases that lack mannose-6-phosphate?

A. Remain in cytosolB. Are degraded by the ERAD systemC. Remain in GolgiD. Are secreted from the cell

Protein sorting in the trans-Golgi network (TGN)

Summary

Secretory and biosynthetic pathway

• Major outward traffic (ER Golgi PM)• ER: protein synthesis, standard glycosylation, quality control by chaperones ( ERAD), synthesis of glycerophospholipids• Golgi: several compartments that differ in ionic conditions. Modification of sugar-tree added in ER, in TGN: protein sorting ( PM, secretory vesicles or lysosomes)• Mannose-6-phosphat serves as sorting signal for lysosomal proteins. M6P-tagged proteins are recognized by the M6P receptor and packaged into vesicles that are delivered to late endosomes• Constitutive secretory pathway: ‘default’ pathway, requires no signals, operates in all cells continously• Regulated secretory pathway: signal dependent, operates only specialized secretory cells

ER Golgi PM

ER

• Synthesis of proteins (rER)

• Disulfid bridges

•Glycosylation of proteins

Golgi

• Modification of sugars

• Sorting of proteins

Overview secretory pathway

•Synthesis of glycero-phospholipids (PS,PE,PC,PI)

•Synthesis of glycolipids

(sphingolipids, glycerolglycolipids)

The major types of lipids in eukaryotic cell membranes

Choline Phosphatidylcholine (PC)Ethanolamine Posphatidylethanolamine (PE)Serine Phosohatidylserine (PS)Inositol Phosphatidylinositol (PI)

Synthesized in ER

Sphingolipids (major type of glycolipid)

Fatty

aci

d

Sph

ingo

sine

headgroupFa

tty a

cid

glycerolP

headgroup

Fatty

aci

d

Glycerophospholipid

No headgroup CeramidePhosphorylcholine or Phosphoroethanolamine SphingomyelinSugars Glycosphingolipids

Synthesized in Golgi

Synthesis of glycerophospholipids occurs at the cytosolic side of the ER

Lipid transporters flip lipids between the layers

Glycolipids are synthesized at the lumenal side of the Golgi membrane

Sphingolipids

Fatty

aci

dS

phin

gosi

ne

headgroup

Ceramide

Asymmetric distribution of lipids in the plasma membrane

Endocytic pathways

Secretory pathways

Pathways of vesicular transport in the cell

Import

Export

Retrieval pathways

Recycling

Vesicular traffic in the cell

1. Secretory/exocytic pathway: major outward traffic (ER Golgi cell surface) 2. Endocytic pathway: major inward traffic: (cell surface endosome lysosome)3. Retrieval pathways (Endosomes Golgi, endosomes cell surface, Golgi ER)

endocytosis

“Uptake of material into the cell by an invaginationof the plasma membrane and its internalization in a membrane-bounded vesicle”

Phagocytosis (“cell eating”),vesicle >250 nm

Receptor-mediated endocytosis:Selective, coated vesicles

Pinocytosis (“cell drinking”) vesicle <150nm

Amoeba catching a ciliate

Protozoa feed by engulfing prey (other protozoa, bacteria)

Phagocytosis is a feeding strategy in protozoa

The lysosome is the cells ‘stomach’.

•Lysosomes are acidic compartments that contain many digestive enzymes (acid hydrolases)•Lysosomal enzymes break down macromolecules and release metabolites into the cytoplasm

Phagocytosis in higher organisms: Cleaning-up

100 Mio red blood cells are ‘eaten’by macrophages per day

Macrophage engulfing two red blood cells.

Phagocytosis in higher organisms: Defence

Macrophage engulfing bacteria

Pinocytosis (“cell drinking”)

• Endocytosis of fluid and small particles.• Continuously active process

A macrophage swallows 25% of its own volume each hour.

90% of its plasma membrane is internalized each 30 min.

Constitutive endocytosisis balanced by contitutive exocytosis

Receptor-mediated endocytosis

Acidic environment:Many receptors loose their cargo

The endosomal compartments

• Complex set of connected tubules and vesicles

• Early endosomes are sorting stations (comparable to TGN) which send vesicles to recycling endosomes or late endosomes

• Recycling endosomes return proteins to the PM (e.g LDL receptor)

• Early endosomes gradually mature into late endosomes by fusion with vesicles or late endosomes

• Late endosomes receive vesicles from the TGN (e.glysosomal proteins)

• Late endosomes send vesicles to the TGN (e.g. recycled M6P receptor)

15.13-endosome_fusion.mov

recyclingendosome

acidity

Cargo receptors may take three different routs in epithelial cells

recyclingendosome

1. Recyclingproteins return to the same plasma membrane domain from which theycame

2. Degradationproteins progress to lysosomes and get degraded

3. Transcytosisproteins can proceed to adifferent domain of the plasma membrane

Vesicular traffic in the cell

1. Secretory/exocytic pathway: major outward traffic (ER Golgi cell surface) 2. Endocytic pathway: major inward traffic: (cell surface endosome lysosome)3. Retrieval pathways (Golgi ER, endosome Golgi, endosome cell surface,)

ER resident proteins are returned to the ER from the Golgi

• KDEL is the C-terminal ER retention signal of ER resident proteins

• The KDEL receptor captures soluble ER resident proteins in the Golgi and carries them back to the ER

• KDEL receptor bind the protein at slightly acidic pH (Golgi)

• KDEL receptor dissociates from protein at neutral pH (ER)

Retrieval pathway: late endosome trans Golgi network

Recycling of cargo receptors between TGN and late lysosomes

Example: M6P receptor

Retrieval pathway: early endosome plasma membrane

Recycling of cargo receptors from endocytotic vesicles

Receptors that dissociate from their cargo in early endosomes

recycling endosomes PM

Example: LDL receptor

Recycling of synaptic vesicles

Summary II

Endocytic pathway• Major inward traffic (PM early endosome late endosome lysosome) • Different types of endocytosis: phagocytosis, pinocytosis ( incl. receptor-mediated endocytosis)• Different function of phagocytosis: feeding (protozoa), cleaning-up and defence (in higher animals, by specialized phagocytotic cells)• Pinocytosis occurs continuously, is mainly carried out by clathrin-coated pits and vesicles that contain cargo receptors (receptor-mediated endocytosis)• All endocytosed material is first delivered to early endosomes/ sorting endosomes ( recycling endosomes or late endosomes )

Retrieval pathways• Recycling cargo receptors from early endosomes to plasma membrane or from late endosomes to TGN• Return of ER resident proteins from Golgi to ER