BIOL2210 Signalling Trafficking and turnover

are there many diff targeting motifs that target proteins for delivery to e.g. mito, peroxisome, nucleus

yes

phagocytosis overview

use pmemb to engulf large particles

form phagosome

pinocytosis

form ruffles

nonspecific uptake of extracellular fluid

req lot of ATP

vesicle formed fuses with lysosome

receptor mediated endocytosis/clatherin-dependent endocytosis

dep on sorting sequence the cargo taken up has, det. where delivered to in cell

e.g. EGFR, LDLR, transferrin receptor

transferrin receptor

binds soluble holo-transferrin (transferrin with bound Fe)

receptor mediated endo/clatherin dep endo

surface expression reg in response to intracellular Fe conc

ein early endosome, low pH causes Fe rel, then transferrin:receptor recycled to memb

examples of clathrin independent endocytosis

macropinocytosis

endocytosis

link between lipid rafts and endocytosis

regulate endocytosis, are often where the pits form!

have diff memb properties, structure, dynamics and signalling

e..g. contain specific receptors that mediate endocytosis of certain material

do some viruses associate with lipid rafts

yes, get endocytosed

EGFR endocytosis at low and high EGF conc

at low conc, clatherin mediated endo

at high conc, clathrin indep endo (receptor get ub)

go to endosome

after EGF dissoc the receptor recycled bac to pmemb if clathrin med, but ub form usually sent to lysosome for degredation

what does lipid raft promote

clustering of receptors (dimerise pot) so promote signalling

can be proximity or ligand induced dimerisation

how do study secretory pathway with VSV-G (protein)

VSV-G is a memb glycoprotein

prod by infected or transfected cell as would secretory protein

use temp sensitive VSV-G to switch on/off its transport

40 oC is misfolded and ER retained

32 oC correctly folded so move thru secretory pathway

req fluorescently tag it tho

how can modifications to VSV-G be used to det. location

proteins leaving ER get glycosylated

Man8(GlcNAc)2 trimmed to Man5(ClcNAc)2 by golgi resident mannosidases

Trimmed proteins are sensitive to endoglycosidase-D cleavage (endo-D) (So ER resident dont get cleaved)

Distinguish forms via size on SDS PAGE

what proteins are suseptible to cleavage by endoglycosidase D (endoD)

mannose trimmed glyosylated proteins

(ones that reach golgi)

can pulse-chase experiments be used to study the secretory pathway

yes, incubate transiently with radioactive aa’s and observe their movement thru pathway

what drives protein coat assembly and therefore vesicle formation

polymerisation of soluble protein complexes on the membrane

provs curvature and acts as a filter (bind specfiic proteins that get incorporated)

req dynamin for budding off

(pit formation often at lipid rafts (specific proteins tend to localise to rafts) and specific lipids give structure, dynamics and function

5 mechanisms of producing membrane curvature

by lipid composition in memb (e.g. PE)

large curved proteins scaffold onto and bend the membrane

insertion of Hphobic a-helice of proteins into leaflet

monomers polymerise on memb

high surface conc of memb-binding proteins leads to crowding and steric pressure that bends the bilayer

which GTPases control formation of clathrin coated, COPI and COPII vesicles

dynamin for clathrin

ARF1 for COPI and clathrin - essential for initiating pol (recruit clathrin and adaptor complexes)

SAR1 for COPII

active in their GTP bound form

what does SAR1 adaptor protein recruit to form COPII vesicles

Sec23/Sec24 complexes

mediating memb deformation and vesicle fission (budding off)

what are SNARE proteins

SNAP (soluble NSF attachment protein) Receptor

#60 members

v (R) or t (Q)-snares

diff ones assoc with diff membranes

why are v and t SNARES called R and T SNARES also

R SNARES contribute R residue

Q snares contrihute Q residue in the snare complexes when form together

what signal does regulated secretory vesicles (e.g. insulin) and synaptic vesicles req to fuse with p memb

Ca2+ cytosol conc increase

does VEGFR2 give ligand specific responses

yes

can bind VEGF-A or VEGF-C

give diff outcomes

but can also get ub and degred (targeted by E2 enzymes) to downreg their signalling

tmeporal vs spatial receptor activation

temporal - timing and duration of activation

spatial - physical location of receptor

what family of GTPase proteins are extensively involved in trafficking events

Rab proteins

inv. in budding, transport, docking and fusion (use GTP hydrolysis) for e.g. fusion

as events req E rel from GTP therefore req rab proteins