Proteins are found in a variety of locations inside and outside the cell.  These proteins are synthesized in the ribosomes.  Now how do they leave these organelles for their specific destrinations?  Let's take a look.

Electron microscopes can reveal structures, but the still shots of the prepared, deceased cells do not tell us about function and other details.  A variety of methods have been devised to further research capabilities in an attempt to solve this problem.

1. autoradiography - "self-revealing" radioactive isotopes are applied to tissue samples which are covered by a thin layer of photographic emulsion.  This is exposed by radiation emanating from the radioisotopes.  Silver grains in the emulsion can be detected under a microscope.  James Jamieson and George Palade put labelled amino acids into cells.  After being digested by enzymes and incorporated for synthesis of new proteins, the endoplasmic reticulum was found to be the site where secretory proteins were made.

The pulse-chase experiment use similar treatment only that the specimen are treated with regular unlabelled amino acids afterwards.  Varying lengths for the application of the "chase" show the pathway for secretory proteins.

2. cell fractionation - cells are homogenized (broken up) and centrifuged into fractions for further analysis.

3. semi-intact cells - cells are "gently blown up" so that holes are poked into them by detergents.  Vesicles can then move out of the cell.

4. genetics - screens are used to detect mutants to better understand the function of gene products.  Yeast are very helpful due to their double mutants.

5. enveloped viruses - protein coats are placed around these viruses so that they can be inserted into pathways for analysis.

6. fusion proteins - this molecular level analysis tests pieces and adds fluorescent tags for tracking.

Alterations to a protein can be occur as cotranslational modifications during translation.  This is found with secretory and integral membrane proteins.  Post-translational modifications occurring after translation is usually found in the mitochondria, peroxisomes, and chloroplasts.  These give informational sequences, protein variation, and the eukaryotic event of genetic engineering.

1) dissulfide bond formation (by SH2 groups on cysteines)
        -occurs in the ER lumen (extracytosolic)
        -found in membrane and secreted proteins
        -cysteine residues stablized by covalent bonds
        -hold tertiary structure subunits
        -catalyzed by protein dissulfide isomerase (PDI), a chaperone protein

2) specific proteolytic cleavages
        -can occur anywhere along pathway (ER --> outside cell) but will occur in a specific place for a specific protein
         for transformation of a precursor into a functional protein
        (eg. hormone ---> hormone, zymogen --->active enzyme, protoxin--->toxin)

3) association of subunits
        -catalyzed by binding protein (BiP), a chaperone, in  the rER lumen

4) glycosylation events
        -covalent addition of carbohydrates to proteins
        -roles:   solubility enhancement (OH and charge groups)
                    protection from degradation (hide from proteases)
                    cell-cell recognition
                    cell-matrix interactions
        -usually occurs on secreted and plasma membrane proteins

"O-linked" (all attached in Golgi)
            -sugars are attached to O of OH group of serine, threonine, and hydroxylysine

"N-linked" (attached in Golgi and ER)
            -sugars are linked to N of NH2 group of asparagine
            -longer, highly variable, but not as variable as O-linked
The basal (core) segment of the carbohydrate chain is put together as a 14 sugar oligosaccharide on a lipid carrier, dolichol phosphate, a hydrophobic molecule of 20 isoprene units.  The oligosaccharide is transferred by oligosaccharyltransferase from the lipid carrier to the nascent polypeptide at the asparagine NH2 as the polypeptide is being translocated into the ER lumen.  Here, the following glycosylation events help in folding and assembly of the proteins to prevent aggregation and resulting degradation.  Modification of the core oligosaccharide in the ER with removal of the terminal glucose residues by glucosidases.  Sugar monomers are added one at a time by membrane bound glycosyltransferases which are found in particular regions of the Golgi for determination of sugar sequence. 

Here are some destinations of proteins made in the two possible ribosomes

Free ribosomes (on outer, cytoplasmic face)                                                      rER ribosomes
out of cell (secretory protein)                                                           cytosol (enzyme of glycolysis, cytoskeleton protein)
in the membrane (integral membrane protein)                                    inner surface of plasma membrane (peripheral protein)
in a compartment of the endomembrane system - ER, Golgi,             nucleus (nuclear protein)
        lysosome, endosome, vesicle, vacuole (soluble protein)             peroxisome, chloroplast, mitochondria (post-translational)

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            Synthesis of secretory, lysosomal, or plant vacuolar protein on membrane-bound ribosomes

1) A polypeptide is synthesized on a free ribosome
2) SIGNAL SEQUENCE of 6 to 20 amino acids emerges and targets its nascent polypeptide to the ER membrane.
3) The GDP bound signal recognition particle (SRP) binds to the signal sequence.  (translation is halted, premature
        folding is prevented)
4) Part of the ribosome acts as a GNEF so that once the SRP has a GTP attached, it can take the signal sequence of the nascent polypeptide and its ribosome to the ER membrane.
5) The SRP interacts with the SRP receptor (both initially GTP bound) while the ribosome interacts with the  translocon, a protein lined membrane channel.  The signal sequence is put into the translocon to open the channel.
6) SRP and its receptor are hydrolyzed and the GDP bound SRP leaves which allows translation to resume.
7) The polypeptide is translocated into the ER lumen.
8) Termination of translation allows the ribosome to leave
9) The membrane channel closes.
10) The N-terminus containing the signal sequence is cleaved by signal peptidase
11) Glycosylation can add carbohydrates by oligosaccharyltransferase.
12) Protein disulfide isomerase (PDI) of the rER lumen can strengthen tertiary structure
13) ER lumen chaperones (like BiP) can move the polypeptide into the ER lumen and promote folding.

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                            Synthesis of integral membrane proteins on membrane-bound ribosomes

A build up of improperly folded proteins in the ER can increase the presence of chaperones or shut down protein synthesis.  A TM receptor kinase in the ER membrane can autophosphorylate to inhibit translation.

Integral membrane proteins are cotranslationally synthesized in the ER membrane.  Hydrophobic transmembrane segments called STOP-TRANSFER SEQUENCES bind to the inside of the translocon.  Cleavage of the signal sequence exposes the free N-termimus to give the polypeptide its topology.  The end with positively charged amino acids usually ends up in the cytosolic side thus orienting the polypeptide.  The uncleaved signal sequence as serve as both the stop-transfer and stop-anchor signal (signal-anchor sequence).  The translocation channel is thought to open along its side and release the transmembrane segment into the lipid bilayer.

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Proteins can be targeted by:
1) signal receptor mechanisms - explained above
2) conformation - a protein could keep moving until it reaches a comfortable environment (ionic, pH, etc.) to obtain final conformation or helix length
3) default pathway
4) retrieval...

ER lumenal resident proteins can "escape" to the Golgi, but a common C-terminal sequence called KDEL (for the amino acid sequence Lys-Asp-Glu-Leu) is recognized by KDEL receptors in the cis Golgi (an uncomfortably higher compartment of pH than the ER).  The KDEL retrieval sequence allows for the anterograde retrieval/return of these stowaways back to the ER.
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In the Golgi membranes, proteins will go through each membrane until it finds the right one or until they find a place that they will fold properly.  The trans face of the Golgi can send proteins in 3 ways:
1) constitutive bulk flow - a continual, default pathway that causes the vesicle to be 5-7 times more concentrated
2) regulated secretion - secretory granules discharge in response to stimuli

3) endosome lysosome - V-class ATPases help lower the pH
A) the lysosomal enzymes are phosphorylated in the cis Golgi cisterna at their mannose sugars from N-linked glycosylation (modification of the sugar into mannose 6-phosphate can serve as signal recognition sequences...)
B) mannose 6-phosphate receptors (MPRs) are integral membrane proteins in clathrin-coated pits of the trans Golgi network
        (TGN) that capture the lysosomal enzymes with such mannose signals inside the TGN lumen
C) the MPRs also have an adaptor-clathrin complex that assembles on the cytosolic surface of the TGN
        membrane.  The outer (cytosolic) surface binds to clathrin scaffolding.
D) Before lysosome fusion, the MPRs dissociate from the lysosomal enzymes located in the vesicles.  The MPRs return to the TGN in uncoated vesicles.

bulk phase endocytosis - nonspecific, random uptake of extracellular fluids along with any molecules in it
receptor mediated endocytosis (RME) - specific uptake of extracellular macromolecules as ligands bound to receptors on the outer surface of the plasma membrane.  Coated pits are regions on the membrane where the receptors gather in bundles.  On the cytoplasmic face, clathrin is a protein that covers the invagination to be by forming a network of hexagon "honeycombs."  This is facilitated by their triskelion structure.  Dynamin, a GTP binding protein, self-assembles in short helical collars around the neck of the invaginated pit.  It is proposed that hydrolysis induces dynamin to cut the invagination off the membrane.  Adapter proteins keep the clathrin and the ligand bound receptors in the right places in relation to the vesicle.  In the cytosol, the clathrin is lost from the vesicle before it fuses to an early endosome.  Usually, low pH in this new compartment will cause the release of the ligands.  The receptors are then shuttled back to the plasma membrane.

Cholesterol is a hydrophobic molecule that is transported in the blood as part of the low-density lipoprotein (LDL).  LDL particles contain a central core of 1500 cholesterol molecules esterified to long-chain fatty acids.  The core is surrounded by a single layer of phospholipids, unesterified cholesterol molecules, and an apolipoprotein B-100 that binds to LDL receptors of cells.  LDL bound to a coated pit will travel by vesicle to lysosomes.  The protein component is degraded and the cholesterol is released for cell use in membranes or steroid formation.

One disease associated with these concepts is familial cholesterolemia.  Leading research on this topic was begun at the University of Texas Medical School in Dallas by Michael Brown and Joseph Goldstein.  The heterozygous condition is bad, but the homozygous is worse.  The four types of problems include:
1) no LDL receptor
2) the LDL receptor is slowly recycled back to the plasma membrane
3) a mutation in the LDL receptor hinders binding
4) LDL  receptors do not cluster (when a cysteine is at position 807 instead of tyrosine)

1. Clathrin coated vesicles - traffic materials from TGN to endosome with the HA-1 adapter
                                            traffic materials from plasma membrane (PM) to endosome with the HA-2 adapter
2. COPI (coat protein) complex vesicles - retrograde movement of materials from Golgi to ER thanks to KDEL
3. COPII vesicles - anterograde movement of materials from ER to cis Golgi

ARF is a GTPase that regulates COPI coating.  ARF must be hydrolyzed before the coat can diassemble.  When ARF is GDP bound, it is cytosolic and its exchange for GTP to become membrane bound is facilitated by a transmembrane protein in the donor membrane that is a GNRP.  A GAP is located on the acceptor membrane.  A GDI and GIP are known.  Brefolden A inhibits the anterograde path. 

90% of proteins in the mitochondria and chloroplasts are imported.

Target sequences:
                            Mitochondria = target sequence at at N-terminus
                            Chloroplast = transit peptide

Energy required for translocation:
A cytosolic chaperone, Hsp70, uses ATP to keep the precursor protein in an unfolded, translocation ready state.  The N-terminus is partite with a signal for getting into the mitochondria and another for getting into the inner mitochondrial memberane.  The contact site where the inner and outer mitochondrial membranes merge have import complexes that allows electrostatic potential to move the positively charged targeting signal into the matrix.  The following is the "conservative sorting" from the prokaryotes of the past which still exist...  In the matrix, chaperones pull the protein through.  ATP is used for
mHsp 70 to help the protein to fold.  The barrel shaped mHsp 70 also uses ATP to partially fold the protein.  A protease cleaves off the N-terminal targeting signal.
Hsp 70 pulls the protein through in an extended form just like in the mitochondrion.
Proteins can contain a stroma-targeting domain or both a stroma-targeting domain and a thylakoid-transfer domain at the N-terminus. 

Targeting Destination (sequence)            Location                Cleaved?                Special Characteristics

1) rER (signal sequence)                             N-terminus                     yes           internal hydrophobic core of 6-12 amino acids

2) mitochondrion uptake (target sequence)   N-terminus                    usually             may be bipartite

3) chloroplast transit peptide (transit sequence)  N-terminus              usually            may be bipartite

4) peroxisome target signal                            C-terminus                    no                SKL (ser-lys-leu)

5) nucleus-nuclear localization sequence (NLS)   usually internal        no                KKKRK, basic
 
 

-about 50% of the cell's proteins are transported into or across a membrance