at NH2 terminus, the signal peptide is the first thing synthesised and all contain one or more positively charged amino acids next to stetch of 6-12 hydrophobic residues

SP are recognised by signal recognition particle (6 proteins, 300 nucleotide RNA)

ribosomes read mRNA in cytosol on free ribosome, then a 16-30 residue ER targeting sequence in new protein directs ribosome to ER and then dock to ER

SRP-protein-ribosome then attaches to ER membrane bound receptor. P54 (a SRP protein) chemically cross-links with ER signal sequence using its M domain (containing many hydrophobuc side chains that form cleft which binds to signal sequence via hydrophobic interactions)

SPs are cleaved from protein by signal peptidases (whose active site faces the lumen), the peptide chian continues to elongate once complete translocon closes and leaves SP in channel

the number of internal hydrophobic transfer sequences determines number of trans membrane domains and the sequences are collectively known as the topogenic sequences

internal signal binds to SRP which binds to SRP receptor which then moves to ribosome and is not cleaved

internal signal sequence binds to SRP which binds to receptor which targets translocon

it is orientated with Cis face towards nucleus and trans face towards the membrane and medial in the middle

proteins synthesised in ER are packed into vesicles and transported to Golgi

glycosylated in different stacks and packed into vesicles and transported to membrane

retrograde transport is Golgi-> Golgi (this is the cisternal progression model) and Golgi-> ER

Golgi-> endosome or plasma membrane->endosome uses clathrin (this controls cell signalling)

phospholipids are resistant to curvature so vesicles are thermodynamically unfavourable so is maintained by coat proteins

Cis-Golgi is formed when COPI vesicles containing vesicles from Cis-Golgi stack fuse with Cis-Golgi network

MEdial-GOlgi is formed when COPI vesicles containing enzymes from medial-Golgi stack fuse with Cis-Golgi network

Trans-Golgi is formed when COPI vesicles containing enzymes from trans-Golgi stack fuse with medial-Golgi network

adaptors (Sec23/24) bind receptors, membrane cargo proteins are recruited to vesicle using the Sec23/Sec24 complex

GTPase hydrolyses GDP to release Sar1 making the coat unstable which allows binding

it is the movement of membrane impermeable molecules into the cell and is controlled by clathrin and adaptors

dietary lipids and cholesterol transported in shell composed of apolipoproteins overlaying

it binds to LDL receptor (839-residue type I membrane glycoprotein with cysteine rich repeats) by apoB-100 protein which causes formation of clathrin coat and endosome

the endosome becomes a lysosome, lysosomal proteases hydrolyse their surface apolipoproteins and lysosome cholesteryl esterases hydrolyse their core cholesteryl esters

mutation in LDL receptor or ApoB causes familial hypercholesterolemia; in heterozygotes LDL in blood doubles and homzygores have increase 4x to 6x. Mutation in Asn-Pro-X-Tyr (the sorting signal that binds to AP2 complex) can cause LDL receptor to be incorporated into coated pits

endocytosis leads to clathrin coats which attaches to membrane via adaptor

it is formed from hexomeric complexes called triskelions, these arrangements give the circular vesicle formation

synamin forms a spiral structure around neck of budding vesicle

GTP hydrolysed which narrows the spiral pulling the membranes closer which excludes water allowing hydrophobic regions to fuse

V-SNARE are incorporated into vesicle membrane during membrane assembly, v-SNAREs then bind to t-SNARE in a tight complex and form a 4 helix bundle

this brings 2 membranes close together in coiled coil complexes form, which excludes water and the fusion is via hemifused intermediate

fundamentally the proteins do not move but the enzymes from other Golgi stacks redefine its characteristic so a cis turns into a medial then into a trans

hydrolysis of bound GTP causes disassembly of SRP and its receptor, and initiates release of bound GDP

These hydrophobic residues form a binding site used for interacting with targeting machinery

The SRP and nascent polypeptide chain-ribosome complex binds to ER at SRP receptor (made of a and B subunits) this interaction is strengthened when GTP bound to P54 and a subunit (FtsY)

movement of nascent chain and ribosome to translocon which opens the channel, so new protein is synthesised through channel into lumen

first identified with mutations of yest protein Sec61alpha (3 other proteins were then found)

consists of 10 transmembrane helices that form a central channel allowing polypeptide passage, helices split into 2 groups of 5

IN first step bundles open to expose hydrophobic binding pocket, SP binds to Sec61a with N terminus facing cytosol

as growing peptide enters SP is cleaved by signal peptidases that recognise sequence on C terminal

all type I proteins contain approximately 22 amino acids that become membrane spanning a helices

Once N terminus enters, SP is cleaved and growing chain continues but when transmembrane domain passes translocon its passage stops and the domain laterally moves into membrane

The hydrophobiciity of this segment anchors it into membrane and is called the stop-transfer anchor sequence

synthesis of polypeptide continues but not through translocon but into cytosol

HGH receptor is typical type I insertion and mutants with charged residue have translocation entirely into ER

possess a single internal hydrophobic signal anchor sequence, in type II this directs insertion of nascent polypeptride into ER membrane so N terminus faces cytosol using type I mechanism

However internal signal anchor sequence is not recognised by signal peptidases so not cleaved, the signal anchor sequence can move laterally into bilayer where it functions as anchor

as elongation continues the C terminal region is extruded through translocon into ER lumen

This also have a single internal hydrobic signal anchor near the N terminus and directs insertion of nascent chain into ER mebrnae with N terminus facing lumen

the signal anchor sequence also functions like a stop transfer sequence and prevents further extrusion of elongating chain into ER lumen

hydrophobic segment close to N terminus is inserted into ER membrane

as nascent chain following transmembrane segment elongates it moves through translocon until second hydrophobic segment is formed

This helix prevents further extension of nascent chain through translocon so function similar to stop transfer anchor sequence

the hydrophobic transmembrane segment closest to N temrinus is often folowed by cluster of positively charged amino acids so nascent polypeptide is inserted into translocon with N terminus extending into lumen

proteins are glycosylated (Asparagin(N)-linked glycosylation makes proteins hydrophilic to stop aggregation and helps folding) (see 13.3)

Begins with oligosaccharide precursor with 14 residues, which is then added to protein

They are then modified by changing up to 9 of the 14 residues

Prior to transfer to polypeptide chain the precursor is assembled on dolichol phosphate (attached by pyrophosphate bond), the 3 glucose residues are the last added during synthesis and act as signal

The 14 residue precurosr is transferred from dolichol to asparagine residue (in Asn-X-Ser and Asn-X-Thr only since needs oligosaccharyl transferase) on nascent polypeptide

Immediately after this transfer glycosidases remove the 3 glucose residues and on mannose residue

They are formed by oxidative linkage of sulfhydryl groups on 2 cystein residues catalysed by protein disulphide isomerase (PDI) (abundance in ER of secretory cells)

Disulphide bonds in PDI active site can be reasily transferred to protein by 2 sequential thiol disulphide transfer reactions which generates a reduced PDI (regenerated by Ero1 which carries a disulphide bond that can be transferred to PDI)

Proinsulin has 3 disulphide binds that link Cys1 and 4, 2 and 6, 3 and 5

BiP can bind transiently to nascent polypeptide chains as they enter the ER during contranslational translocation

calnexin and calreticulin selectively bind to some N-linked oligosaccharides on growing chains (its ligand formed by glucosyltransferase), binding of these 2 to unfolded chains marked with glucosylated N-linked oligosaccharides prevents aggregation

Peptidyl-prolyl isomerases accelerate rotation about peptidyl-prolyl bonds at proline residues in unfolded segments

Ire1 is an ER membrane protein that exists both as a monomer and dimer

this is the stress response that responds to accumulation of unfolded protein in ER by activating enzymes involved in protein folding

the dimeric form promotes Hac1 formation that activates expression of genes induced in this response, but the dimer formation can be prevented by BiP

Another response to unfolded proteins is proteolysis of ATF6 (transmembrane in ER), its cytosolic domain is released by proteolysis and moves to nucleus to promote chaperone transcription through Notch signalling pathway

Studeis have shown that misfolded secretory proteins are recognised byspecific ER membrane proteins and are targeted for transport from ER lumen into the cytosol by dislocation

recognition involves trimming of Nlinked carbohydrate chain by mannosidases in Er which are recognised by OS-9, those with no oligosaccharide can be targeted suggesting other processes

ERAD complex enables dislocation of misfolded protein, likely by Sec61 translocation channel repurposed

as segments of dislocated polypeptide emerge into cytosol p97 uses ATP to pull into cytosol where ubiquitin ligase of ERAD add ubiquitin

leucine-rich sequence found in PKI adn in the Rev protein of HIV

Other 2 recognised in different heterogenous ribonucleoprotein particles

They are a nuclear-export signal that stimulates their export from the nucleus to teh cytoplasm

a specific nuclear transport receptor (exportin 1) forms complex with RanGTP in nucleus and then binds the NES in a cargo protein

binding of exportin 1 to RanGTP causes change in exportin 1 that increases affinity for NES so a trimolecular cargo complex forms

exportin 1 then transiently interacts with FG repeats in FG nucleoporin and diffuses through NPC

cargo complex dissociates when it encounters the RanGAP associated with NPC cytoplasmic filaments stimulating hydrolysis of bound GTP which lowers exportin 1 affinity

RanGDP dissociates from trimolecular cargo complex, exportin lowers affinity for NES whihc releases cargo into cytosol

exportin 1 and RanGDP are then transported back into the nucleus through the NPC

once completely processed, mRNA remains associated with proteins in a messenger ribonuclear protein complex (mRNP)

principal transporter of mRNPs is the mRNP exporter (composed of nuclear export factor 1 and nuclear export transporter1), multiple of NXF1/NXT1 dimers bind to mRNPs through cooperative binding

both NXF1 and NFT1 interact with FG repeats which allows diffusion through the central channel of the NPC

Once mRNP-NXF1/NXT1 complex reaches cytosol the dimers dissociate from mRNP using RNA helicase Dbp5

Sar1 binds to GTP and triggers COPII vesicle coat assembly (Sar1 is also only GTPase switch protein activated in ER membrane so only COPUU bud from ER)

Sec12acts as a GEF for Sar1 so GDP is released and binds GTP, this GTP binding causes change in Sar1 which exposes amphipathic N terminus which embeds itself in bilayer

After vesicle coat formed, Sec23 coat subunit promotes GTP hydrolysis by Sar1 and release of SarGDP from vesicle membrane disassembles coat

ARF protein is a GTP binding protein and undergoes similar nucleotide exchange and hydrolysis

myristate anchor (covalent modification) on N terminus of ARF tethers it ARFGDP to Golgi which changes ARF conformation

The change means hydrophobic residues in N terminal segments insert into membrane bilayer and the tight association allows coat assembly

Rab proteins contain isoprenoid anchor allowing them to be tethered vesicle membrane

cytosolic RabGDP is targeted to vesicle using sorting signals on vesicle for identification, and attach by insertion of the anchor into vesicle membrane facilitated by guanine nucleotide dissociation inhibitor

a specific GEF in vesicle membrane converts RabGDP to RabGTP and when activated RabGTP can bind to Rab effectors

this binding allows docking and after fusion the GTP on Rab is hydrolysed releasing RabGDP

Following membrane fusion NSF binds to SNARE complexes, adn ATP hydrolysis drives dissociation of SNARE complexes so can complete cycle (this occurs simultaneously with RabGTP hydrolysis)

Example- Sec4 tags vesicles and Sec4GDP binds where it is actived to Sec4GTP by GEF which then binds to receptor (called exocyst)

VAMP (the v-SNARE) is incorporated into membraen as bud from trans-Golgi network, and syntaxin and SNAP-25 (t-SNAREs) are attached to membrane by lipid anchor

The 4 helix bundle forms as 1 from VAMP, 1 from syntaxin adn 2 from SNAP-25 coil around each other

the embedded hydrophobic transmembrane domains of VAMP and syntaxin pull the vesicle to target membrane

the energetically favourable formation of 4 helix bundle can overcome electrostatic repulsion of negative phospholipid heads allowing fusion

COPII formation triggered when Sec12 exchnages bound GDP for GTP in Sar1, this exchange induces Sar1 binding to ER binding and to Sec23/Sec24, this then allows binding of Sec13/Sec31 to form COPII

Sec13/Sec31 spontaneously assemble into cagelike structures

Sec16 (ER cytosolic protein) interacts with Sar1GTP to organise coat proteins and increase polymerisation efficiency

ER membranes can be recruited to COPII, many containing di-acidic sorting signals whihc bind to Sec24 on COPII coat and are involved in selective export of certain proteins from ER

COPI coat is formed from coaromers (cytosolic complexes made of 7 polypeptide subunits

Most ER resident proteins carry Lys-Asp-Glu-Leu sequence at C terminus (this is necessary for protein location by the KDEL receptor (a transmembrane protein found in vesicles between ER and cis-Golgi and cis Golgi membrane))

The KDEL receptor and other membrane proteins are transported back to ER using Lys-Lys-X-X sequence that binds to COPa and COPB

cisternae compartments fidder from one another based on the enzymes they contain, and it is now known that the small vesicles in the vicinity of the Golgi are mivigng in the retrograde direction so Golgi has a highly dynamic structure

microscopic analysis of algae scales synthesis revealed that they are assembled from glycoproteins in cis into large dense complexes that then move to trans, but were never obsevered in vesicles

IN fibroblasts large procollagen precursors often form in cis lumen bjt are too large to be incorporated into vesicles

fluorescent labels of cis and trans Golgi resident proteins revealed that few compartments ever contained both proteins but over time cis Golgi progressively lose this protein and acquire trans

The best characterised vesicle contains outer clathrin layer and adaptor protein complex inner layer

the APs determine which cargo are included in budding transport vesicles by binding to cytosolic face of membrane proteins and all coats containing an AP complex uses ARF to initate assembly

when going to lysosome, the clathrin coat is assembled with AP1 or GGA. AP1 binds to proteins with Tyr-X-X-Φsequence, and GGA binds to Asp-X-Leu-Leu and Asp-Phe-Gly-X-Φ

some contain AP3 complex which has no clathrin binding site, they still transport to lysosome but bypass late endosome and fuse directly

dynamin polymerises arounf the neck of the bud and then hydrolyses GTP (which provides energy to drive conformational change)

cytosolic Hsp70 (chaperone) uses energy from ATP to drive depolymerisation of clathrin coat into triskeletons and exposes v-SNAREs for use in fusion with target membranes, cytosolic Hsp70 uses auxillin (co-chaperone)that stimulates ATP hydrolysis

mannose 6-phosphate (M6P) is formed in cis-Golgi and is sorting signal to direct lysosomal enzymes from trans to late endosome

In cis N-linked oligosaccharide on most lysosomal enzymes undergoes process to generate M6P residues on oligosaccharide chains whihc prevents further processing

transmembrane M6P receptors bind the M6P residues on lysosome destined protein, Clathrin/AP1 vesicles (containing this receptor) then bud from trans Golgi, lose their coat and fuse with late endosome

A phosphatase within late endosome usually removes phosphate from M6P on lysosomal enzymes preventing rebinding to M6P receptor adn retromer recycle the receptor back into trans

some only have N-terminal ER signal peptides removed from nascent chain but some membrane proteins and soluble secretory proteins are synthesised as precursors (called proproteins)

proproteins require further processing to generate mature, active proteins and this conversion usually occurs after being sorted into vesicles

proenzymes are sorted by M6P receptor and have delayed activation since they would otherwise digest other components in secretory pathway

proproteins of most constitutively secreted proteins are cleaved only once at a site C-terminal to a dibasic recognition sequence like Arg-Arg or Lys-Arg by endoprotease (examples- furin processes albumin, PC2 and PC3 processes hormones)

In one mechanism the sorting takes place in trans network, the different vesicel types for apical and basolateral can be distinguished by protein constituents (like Rab adn v-SNARE)

mutational studies on VSV G protein that are specifically targeted to basolateral have defined tyr-X-X-Φ and Asp-X-Leu-Leu sorting signals which are recruited into clathrin/AP coated vesicles so clathrin coated vesicles are involved in basolateral membrane

GPI cells are usually targeted to apical membrane, however GPI anchor is not an apical sorting signal in polarised cells and can go to basolateral in thyroid

Another mechanism for sorting apical and basolateral proteins acts in hepatocytes. Both types are transported in vesicels from trans to basolateral by exocytosis

From there both basolateral and apical are endocytosed in the same vesicles but then paths diverge (basolateral sorted into vesicle adn recycled back into basolateral, and apical are sorted into vesicles and move across cell by transcytosi)

LDL is typically sphere, 20-25nm diameter, amphipathic outer layer composed og phospholipid monolayer adn single large apoB-100 protein

The unesterified cholesterol is then free to leave lysosome and used in cell synthesis

at endosomal pH (5-5.5) His residues in B-propeller domain of receptor become protanted causign high affinity to the Cys repeats in LDL binding domain

this causes release of bound LDL particles and the LDL receptor is efficiently recycled back to the plasma membrane