Cell Bio Exam 3

Endocytosis

Default Pathway Transport

Proteins from the ER move along this vesicular transport route (rough ER to Golgi to PM)

Default Pathway Departure

requires specific protein signals; retention and diversion

Critical role of the Golgi Body

biosynthesis, sorting, dispatching, and recycling to various parts of the cell

Golgi function

remove and add sugars to glycoproteins through the action of resident glycosidases and glycosyltransferases

cis-Golgi network

passes proteins to the stack or returns them back to ER

trans-Golgi network

passes proteins to the plasma membrane, lysosomes, or secretory vesicles

Rate limiting step in protein transport and secretion

ER to golgi

Quality control step: Protein correctly assembled

then protein is exported to the cis-Golgi network by the default pathway

Quality control step: Protein not correctly folded

Denatured or unfolded protein is degraded (can be detrimental like CFTR degradation)

Quality control step: Protein not correctly assembled with other subunits

If still bound to BiP or other chaperones and is actively still undergoing folding, then protein remains in the ER

ER proteins that are retained

BiP and PDI

ER and cis-Golgi receptors

recycle KDEL containing proteins in the cis-Golgi by ferrying them back to ER via transport vesicles

ER KDEL retention sequence removal

results in protein transport to the cell surface via the default pathway

KDEL sequence fusion

to non-ER proteins will retain them in the ER

KDEL sequence makeup

short-stretch of aa sequence (Lys-Asp-Glu-Leu)

ER proteins are either

resident or en route to other destinations

ER proteins before leaving RER must be

folded, modified, and assembled correctly for proper function

RER protein chaperones are

BiP (hsp70 member), calnexin, calreticulin

RER protein chaperones function

prevent aggregation of hydrophobic domains, facilitates folding in ATP-dependent manner, and helps retain partially folded proteins in the ER avoiding premature transit to the Golgi

RER isomerases

protein disulfide isomerase (PDI)

Glycoproteins

All proteins that are exposed to the ER lumen and post-translationally glycosylated

Glycosylation: additions to protein

prefabricated oligosaccharide complex is added en bloc to specific sites on protein chain

Oligosaccharide contains sugars

14= 2GlcNac, 9 Man, 3 Glu

Glycosylation sequence

Asn-X-Ser/Thr, oligosaccharide attached by N-glycosidic bond to Asn side-chain (N-linked or Asn-linked)

Oligosaccharide assembly via transfer reactions

Assembled from nucleotide-and lipid-phosphate linked monosaccharides in the cytosol and ER through a sequential series of transfer reactions

Oligosaccharide assembly carrier lipid

dolichol-PO4 aka polyisoprenoid

Oligosaccharide assembly sugar donors

UDP-GlcNAc, GDP-Man, Dol-P-Man, and DOL-P-Glc

Oligosaccharide assembly modifications

following partial assembly of (GlcNAc)2(Man)5 on the cytosolic side, lipid-sugar precursor flips to lumenal side of the ER for final modifications

Protein Glycosylation Reaction catalyst

oligosaccharyl transferase

Oligosacchryl Transferase function

mediates the transfer of oligosaccharide from lipid carrier to the Asn resiude on the protein

Protein Glycosylation Reaction: Association

Oligosaccharyl transferase is closely associated with the SEC61 complex so protein modifications occur relatively quickly following translocation into the lumen

Glycan Trimming ER

Original high-mannose glycan complex is further processed and terminal 3-Glc and 1-Man is removed before exiting the ER

Glycan Trimming Golgi

further removal and addition of sugars occurs, glycosidases remove and glycosyltransferases add

Sugar processing in the golgi involves

complex oligosaccharides and high manose oligosaccharides

cis Golgi network sorting

involves phosphorylation of oligosaccharides on lysosomal proteins

cis cisterna of Golgi stack function

removal of Man

medial cisterna of Golgi stack function

removal of Man and addition of GlcNAc

trans cisterna of Golgi stack function

addition of Gal and NANA

trans Golgi network sulfation

of tyrosines and carbohydrates, then sorting occurs

Golgi vesicular transport model

Golgi is static and transport vesicles ferry cargo between stacks

Cisternal Maturation Model

Golgi cisternae mature as they move forward through the stack. At each stage Golgi resident proteins carried forward are moved backward via vesicle transport

Lysosome acid hydrolases

nucleases, proteases, glycosidases, lipases, phosphatases, sulfatases, phospholipases

the lysosomes is the site where

several distinct vesicular pathways converge

Lysosomal pathways

biosynthetic (ER-Golgi), endocytic, autophagic, and phagocytic pathway (macrophages and neutrophils)

Lysosomal hydrolase synthesis

hydrolases and membrane proteins are synthesized and sorted by Golgi and then delivered via transport vesicles that traffic through late endosomes

Lysosome signal patch

read by glycosyltransferases in cis-Golgi that add mannose 6-phosphate (Man-6-P) tag to proenzymes which is read by receptors present in the trans-Golgi network

Man-6-P receptor functions

binds and segregates hydrolases from other other protein traffic exiting the Golgi

Vesicle forward transport

ER phosphate addition to M6P receptor binding to Receptor-dependent transport

Vesicle recycling transport

Receptor dependent transport to lysosomal phosphate removal to acidic pH disassociation to recycling to Golgi

Lysome-associated membrane proteins

LAMPs follow default secretory pathway to the plasma membrane

Lysosome Membrane Protein Pathway

default pathway to plasma membrane also followed by small percent of hydrolases and Man-6P-R. Scavenger pathway elucidated by certain lysosomal storage diseases

Hurler Disease basis

lysosomal enzyme required for the breakdown of glycosaminoglycans (ECM proteins) is missing; large accumulation of undigested material

Hurler Disease rescue

diseased culture cells are co-cultured with normal cells, which rescues mutant cells and is capable of breaking down GAGs

Hurler Cell scavenging

normal cells produce missing hydrolase and some escape via default pathway. Hurler cells can scavenge WT hydrolases in media using normal Man-6-P receptors at PM

I-Cell disease

hydrolases are missing due to defect in GlcNAc phosphotransferase so there is no Man-6-P tag, receptor fail to sort and target hydrolases

Reasons for endocytosis

uptake of nutrient molecules; clearance of harmful substances, cellular debris, and infectious organisms; maintenance of surface-to-volume ratio

The type of endocytosis is distinguished

by the size of the import vesicle and the material being ingested

Phagocytosis

greater than 250nm; uptake of large, particulate matter

Receptor-mediated endocytosis

between 250-300 nm; uptake of macromolecules via specific cell surface receptors

Pinocytosis (cellular sipping)

less than 150 nm; uptake of fluid and dissolved solutes

Much of the ingested material is delivered to

lysosomes

Protozoa

does phagocytosis, ingestion of food particles

Macrophages and neutrophils

phagocytes that help with binding, clearance, and destruction of bacteria, damaged cells, and cellular debris

Fibroblasts

phagocyte that helps with connective tissue remodeling

Receptor-mediated event

cells possess surface receptors that recognize specific or generic sites on various particles they then bind particle the progressively extend pseudopodia to engulf particle

Engulfment requires

receptor-ligand interactions around the surface of the particle

Receptor-ligand interactions induce

localized changes in the cortical cytoplasm

Calcium fluxes at cortical cytoplasm

induce formation of gel-like cytoplasm

Pseudopodia receptor-ligand interactions

membrane-coupled pseudopod extension and actin polymerization

When is engulfment at pseudopodia completed

when pseudopodia meet and fuse forming phagosome

Ingested particles initially reside

in phagosomes where oxygen metabolites help kill bacteria

Lysosomes fuse with phagosomes

to form phagolysosomes that have acid hydrolases to digest phagosome contents

Residual bodies

phagolysosomes that contain non-digestible material

Pathogen escape mechanism

Free bacterium enters host cell via zipepr mechanism

Zipper mechanism

Bacterium’s adhesin attaches to receptors (integrins, cadherins) on host cell

Trigger mechanism

Bacterium attaches to type 3 secretion apparatus

Pathogens can also invade

non-phagocytic cells

Yersinia

invasin-beta1 integrins (zipper)

Listeria

E-cadherin (zipper)

Salmonella

effector injection in host cell (trigger)

Virulence methods used by pathogens

1. escape

2. prevent fusion with lysosomes

3. survive in phagolysosome

Listeria escape mechanism

Receptor-mediated nutrient endocytosis

Cholesterol uptake via LDL receptors, delivery to lysosomes (lipoproteins like LDL, HDL and metals like Fe)

Brown and Goldstein won the nobel prize for

Genetic analysis of human disposed to heat attacks due to atheroscloerosis, defective LDL receptors, resulting in increases in blood serum cholesterol levels

Hormones, growth factors, and immunoglobulins are uptaken by

receptor-mediated endocytosis

Receptor mediated endocytosis of harmful substances

modified glycoconjugates and coagulation factors

Receptor mediated endocytosis efficiency

is increased to greater than 1000 fold by specific cell surface receptor mediation that bind ligand with high affinity

Receptor-ligand complexed enter cells

specialized membrane structures like clathrin-coated plaques and pits

Clathrin coated pits

specialized endocytic structures that collect endocytic receptors, excluding other membrane proteins and concentrating endocytic receptors

Defective receptors in clathrin coated pits

reduced binding to cargo LDL and have defective cytoplasmic tails

Clathrin coat proteins

clathrin heavy and light chains along with adaptor complexes that mediate interaction between receptors and clathrin

Clathrin assembly drives

coated pit formation and vesicle budding

Clathrin heavy chains

180 kDa hinged rods

Clathrin light chains

33 kDa accessory proteins

Clathrin protein structures

triskelion, formed of heterohexamer (3 heavy aand 3 light chains)

Triskelia polymer formation is regulated by

clathrin light chains

Triskelia polymerization

the self-assembly of clathrin triskelions into a lattice-like polymer network which is composed of hexagons and pentagons

Triskelia polymerization assembly process acts

to mechanically form the curved, budded membrane

Clathrin adaptors

aka adaptin, complexes that mediate clathrin binding to specific membrane receptors

Clathrin PM adaptor

AP-2