Post-Golgi biosynthetic pathways

MDCK-cell Resting fibroblast Migrating fibrobl.

The epitelial cell line MDCK is the most studied model system for polarised sorting and transport.

Hepatocyte Retinal pigment Retinal rod cell

epitelial cell

Hippocampus

neuron

Osteoclast Budding yeast cell

Sorting along the biosynthetic pathway in epitelial cells. MDCK-cells as model system.

Sorting in the trans-Golgi network.

THE SORTING DEPENDS ON SIGNALS IN THE MOLECULES TO BE SORTED

Classical signals:

*Sorting of lysosomal enzymes to lysosomes

*Basolateral transport in epithelial cells.

*Retrograde transport from the Golgi to ER.

*Endocytosis of receptors and other molecules from the cell surface – a fraction is sorted to the trans-Golgi network.

Before any basolateral sorting signals were identified (1991 ->), it was suggested that basolateral transport occured by “bulk flow” while transport to the apicale side – which is the specialised domain in epithelial cells – would require sorting.

In 1991 it was published, however, that the transmembrane protein pIgA receptor was transported basolaterally in a signal dependent manner.

Growth of MDCK epitelial cells on filters. Transfer to glass-dishes with 90 ml of medium for establishment of confluent cell layers.

Basolateralt medium

Apikalt medium

PROTEIN SORTING STUDIES IN THE SECRETORY PATHWAY OF EPITHELIAL CELLS

A protein transported equally well to the apical or basolateral surfaces is regarded as a protein without a sorting signal.

Secretory proteins are the ones most easily studied, because these may be identified after harvest of the two opposite media of filter-grown cells.

Apical or basolateral membrane proteins may be extracted after differetial biotinylation of the two opposite sides and/or recognition by specific antibodies.

Sorting signals are autonomous – they work after ”transplantation” to normally non-sorted molecules.

Baso

Api

Baso

Api

Api

Api

Baso

Api

Lumenal domain tm

Cytoplasmic domain

Golgi membrane /endosomal membrane

Cytoplasm

Lumen

Cytoplasmic domain

Transmembrane domain

Lumenal domain

Golgi membrane /endosomal membrane

Cytoplasmic domain

TM domain

Lumenal domain

Truncated variant

Example: Polymeric IgA receptor = pIgR

Basolateral

Apical

Golgi membrane /endosomal membrane

Cytoplasmic domain

TM domain

Lumenal domain

Truncated variant

Example: Polymeric IgA receptor = pIgR

Basolateral

Apical

3 aa

Golgi membrane /endosomal membrane

Cytoplasmic domain

TM domain

Lumenal domain

Example: Polymeric IgA receptor = pIgR

Basolateral

Apical

Recessive apical sorting information

Golgi membrane /endosomal membrane

Cytoplasmic domain

TM domain

Lumenal domain

Deletion of amino acids 655-668 = 14 amino acids

Basolateral

Basolateral

Apical

17 aa

Golgi membrane /endosomal membrane

Cytoplasmic domain

TM domain

Lumenal domain

17 amino acids: RARHRRNVDRVSIGSYR, red identified by ala scan

Basolateral

Baso

Apical

Golgi membrane /endosomal membrane

TM domain

Lumenal domain

ApicalBaso

Apical

Reporter protein: PLAP (placental alkaline phosphatase) extracellular (lumenal) domain

WHAT MOTIFS ARE BASOLATERAL SORTING SIGNALS?

YXX (YVEL/YTDI/YXRF) bulky/hydrophobic TGN38, M6PR

LL / IL / LEL Fc Receptor, MHC II Invariant chain, EGF receptor

NPXY Low density lipoprotein (LDL) receptor

H/R-XXV Polymeric IgA receptor, Mannose-6-phosphate rec. (?)

PXXP Epidermal growth factor (EGF) receptor

Other Transferrin receptor (VDGDNSHVEMKLA)

Some proteins have several sorting signals in their cytoplasmic tail. These are recognized at different intracellular sites, to provide recycling to the basolateral domain.

BASOLATERAL SORTING SIGNALS

Some basolateral sorting signals overlap with endocytosis signals.

*Fc receptor

*Asialoglycoprotein receptor

*Lysosomal acid phosphatase

Other basolateral sorting signals are distinct from endocytosis signals.

*Polymeric IgA receptor

*LDL receptor

*Transferrin recptor (?)

HOW AND WHERE IS BASOLATERAL SORTING INFORMATION RECOGNIZED?

Adaptor complexes 1-3 Lysosomal route Basolateral route

via endosomes

The first proteins regarded as mediators of basolateral sorting were adaptins – already known to be involved in endocytosis from clathrin coated pits at the cell surface.

The adaptins consist of 2 large, 1 medium and 2 small subunits.

4 different adaptin-complexes have been discovered.

AP-1A: TGN (+ endosomes) AP-1B: Epithelia specific AP-3A: Endosome/TGN AP-3B: Neuron-specific (endosome?)

A/B

Baso

Baso

AP-1, AP-2 and maybe AP-3 (in mammals) may bind clathrin.

All 4 complexes are found in Arabidopsis, but only AP 1-3 in Drosophila.

Many of the subunits are found as closely related isoformes coded by separate genes making a large number of combinations possible.

Endocytosis

AP-1B contains a specific 1B subunit which only is expressed in certain polarised cells (not all polarised cell types, mainly epithelia).

Recognizes tyr-based signals.

AP-4 has also been connected to basolateral sorting, but has equal or overlapping specificity with AP-1B.

There is still room for more adaptors for basolateral sorting.

AP-4

Somewhere in the picture: FAPP1 and FAPP2, mediating TGN => PM transport.

FAPP2

GGA (1-3): Golgi-associated, -adaptin homologous, ARF-interacting proteins

N-terminal hydrofobicsequence

ARF-1

GDP

GEF

AP-1 What about tyr-signals?

Ubq

Rabaptin 5 binding

Ear = GAE

-synergin?

Dynamin

Cortactin

Actin

Membrane

Arp 2/3 complex

At least 6 ARFs exist in mammals, 5 are localised to the Golgi-apparatus and 1 to the plasma membrane.

Aktiv membranbundet

cytoplasmatisk

4 families

GEFs with several members

ARF1-GTP (myristoylated), a tyrosine based signal, and phosfatidylinositol 4,5 bisphosphate are necessary to recruit AP-1 clathrin adaptors to membranes.

Phosphatidylinositides of the 4-series has been regarded as important for Golgi.

GGA dependent receptors

CI-Mannose-6-phosphate receptor

CD-Mannose-6-phosphate receptor

Sortilin

SorLA/LR11

LRP-3

-secretase

Clathrin-dependent

Non-clathrin dependent

Some transmembrane proteins have cytoplasmic domains that interact directly with microtubule motors

What about

APICAL SORTING IN EPITHELIAL CELLS?

Glycans: N-glycans, O-glycans, CS glycosaminoglycans

Yes (maybe and no), yes (maybe), yes.

GPI-anchors? NOT REALLY – OR?

LIPID DOMAINS??

Protein motifs for apical sorting: Megalin NPXY. The second of three NPXY motifs is crucial (distance from membrane…).

MDCK cells transfected with the gene for the non-glycosylated protein rat growth hormone (rGH) secretes this protein randomly, which is slightly more basolaterally

rGH with 2 N-linked glycosylation sites is secreted almost exclusively into the apical medium.

Erythropoietin – three N-glycans, one is critical.

Endolyn – eight N-glycans, not all equally important.

O-glycans of mucin type may also mediate apical sorting:

Intestinal sucrase-isomaltase

Gp-40

Several other examples

But several examples of non-sorted glycoproteins also exist.

HS

CS

Transmembrane lectin-molecule

raft

VIP 36?A

HypothesisDetergent insoluble proteins of apical transport vesicles were separated by 2D-gel analysis and sequenced. One putative lectin molecule was found: VIP 36.

WHAT IS A (GLYCOLIPID) RAFT?

Glycolipid- and cholesterol rich domains in a lipid membrane are associated in a more stable structure than lipids are according to

“the fluid mosaic model.”

On the cell surface of a “regular” cell, these domains will have a diameter of 60 - 100 nm. In specialised membranes may larger areas of the plasma membrane have raft-characteristics.

Example: The apical membrane of epithelial cells (MDCK).

Do lipids and lipid-binding proteins play a role in sorting of molecules that are transported from the TGN to the apical membrane?

Caveolins: Proteins with affinity for specialised lipid-domains. Palmitoylation. Might be necessary for cell surface transport of GPI-anchored proteins. Not apical transport.

GPI-anker: Glycosyl-phosphatidyl-inositol-anchor that might bind proteins to a membrane. For some time regarded as sorting signals for apical transport, since these proteins usually are localised to rafts. The apical sorting is most likely dependent on N-glycans (via transcytosis?).

Glycosphingolipids: are glycolipids that are mainly transported to the apical side in MDCK-cells (from the TGN). Present in rafts rich in cholesterol.

MAL (VIP 17): A protein that seems to mediate apical sorting of several cargo proteins in MDCK-cells.

There are probably several independent transport mechanisms operating in parallell, both to the apical and to the basolateral side of MDCK epitelceller. The apical ones may be raft-based or not raft-based.

Annexin II, Annexin XIIIb, FAPP1/2, MAL (VIP17)

Protein kinase D

All mechanisms for sorting from the TGN are not known

*We have only discussed proteins with one transmembrane domain, while many proteins span the membrane several times. These may also be sorted. How?

*Some apical proteins, like megalin, have been reported to have signals in the cytoplasmic tail (interaction with motor proteins?)

*Ubiquitinylation may shift the sorting from TGN to the plasma membrane towards TGN to lysosomes (the vacuole in yeast).

*Lipids may play a role in sorting in many ways.

*What factors are necessary for budding, transport and fusion?

(v- and t- SNARES, GTP-binding proteins, etc.)