Cell Molec chapter 13&14.txt

Proteins destined to be released (secreted) from the cell. Proteins destined to remain in the lumen of the ER, Golgi, and lysosomes

mRNA binds to Free Ribosomes and translation begins as normal.

-Signal Sequence = The first 16-30 amino acids of proteins destined for the above locations that serve as a binding site for a protein complex called the Signal Recognition Particle (SRP) -Signal sequences usually have stretches of Hydrophobic amino acids (important for binding to SRP) -This binding halts translation (for the time being)

SRP is targeted to the ER because of its attraction for the SRP Receptor -The SRP receptor is a protein complex that is embedded in the ER membrane -SRP will interact with the SRP receptor and bring the ribosome, mRNA, and growing polypeptide along for the ride

ER channel Protein called the translocon to the complex -SRP/SRP receptor then transfers the ribosome (+mRNA/polypep) to the translocon -This requires GTP hydrolysis and causes SRP to fall off from the receptor -This transfer causes the translocon channel to open up, allowing the signal sequence and rest of growing polypeptide to move into the channel

Elongation commences and the growing polypeptide moves through the translocon -The ribosome pushes the growing polypeptide into the channel -While this is occurring, an enzyme located in the ER called Signal Peptidase chops off the signal peptide from the front of the protein

The process of moving a protein across the ER membrane DURING translation is called Co-Translation translocation

Proteins are shipped to their destination within Vesicles -Proteins within the ER lumen will either stay there (if they are meant to) or they will be shipped to their final destination in membrane-enclosed vesicles

Type I proteins= Contain an N-terminal Signal sequence and an internal hydrophobic sequence that serves as the Transmembrane domain -Steps of translocation: a) First steps of cotranslational translocation are the same as for secretoryproteins b) These proteins contain a patch of ~22 consecutive hydrophobic a.a. -The extreme hydrophobicityof this patch causes it to Leave the translocon and move into the lipid bilayer -This hydrophobic patch is often called a Stop-Transfer Anchor sequence c) Elongation continues, but NOT through the translocon Type II/III proteins= Do not contain an N-terminal signal sequence -Instead, they have an internal hydrophobic sequence that BOTH Targets the ribosome to an ER transloconand serves as the Transmembrane domain -Called a signal-anchor sequence -Steps of translocation: a) The polypeptide is made on a free ribosome until reaching the hydrophobic signal-anchor sequence b) This sequence signals the ribosome to find an ER translocon -Binding of the ribosome to the translocon opens it up and the signal anchor sequence enters into the channel c) The extreme hydrophobicityof this sequence causes it to leave the translocon and move into the lipid bilayer�elongation continues through the channel. 2) Type II/III proteins -The 2 types differ based on where their N and C- termini are with respect to the cytosol/lumen 3) Type IV proteins(multipass) -These proteins have both a stop transfer anchor sequence and several signal-anchor sequences -Uses similar mechanisms of membrane entry as described for Type I, II, and III proteins

Patch of 22 hydrophobic amino acids that causes it to leave the translocon and move into the lipid bilayer. Type II/III proteins= Do not contain an N-terminal signal sequence -Instead, they have an internal hydrophobic sequence that BOTH targets the ribosome to an ER transloconand serves as the transmembrane domain -Called a

-Several ER-specific chaperones help to fold polypeptides: a) BiP �Molecular chaperone that binds to new made polypeptides and prevents them from misfolding b) Protein disulfide isomerase(PDI)�This enzyme catalyzes an oxidation reaction that results in the formation of a Disulfide bond (between 2 cysteines) c) Calnexinand calreticulin�Both are Lectins (bind to carbohydrates on glycoproteins) that perform a similar function as BiP

GLYCOSYLATION- This is the addition of a large sugar complex to an asparagine, serine, or threonine within an ER protein. -If added to the Asn, called an N-LINKED oligosaccharide -If added to a Ser/Thr, called an O-LINKED oligosaccaride.

Recent evidence suggests that misfoldedER proteins are unfolded and shipped back out of the transloconin the reverse direction -Called RETROTRANSLOCATION. Neurodegenerative diseases (CJD, Parkinson�s, Alzheimer�s) are thought to be caused in part by defective folding and/or retrotranslocation

Proteins destined for the mitochondria are produced on free (cytosolic) ribosomes and folded after entering into the mitochondria � There are 4 possible mitochondrial destinations (and proteins get to each destination using slightly different mechanisms) 1) In the matrix 2) Embedded in inner mitochondrial membrane 3) In the intermembranespace 4) Embedded in outer mitochondrial membrane

Getting into the mitochondrial MATRIX: 1) Polypeptides are made on free ribosomes -Matrix-targeting sequencethat consists of an AMPHIPATHIC ahelix (+ on 1 side, hydrophobic on the other) is found at the N-terminus of the polypep. 2) MTS is recognized by a IMPORT RECEPTOR located on the surface of the outer mitochondrial membrane 3) The import receptor transfers the polypepto a channel complex that is composed off the protein TOM 40 and is called the general import pore 4) Polypeptide is translocatedthrough this outer membrane pore and then immediately through another pore on the inner membrane (made of proteins called Tim 23 & Tim 17) -Double translocation is possible only when the 2 membranes are close together 5) Upon arriving in the matrix, the targeting sequence is cut off and the polypepis folded properly

-tom 40 is called the general import pore, rotein Tom40 is the main component of the outer membrane translocation machinery for mitochondrial preproteins.

-the inner membrane is made out of tim 17 and 23. The two membranes makes translocation possible when they are both close together

-upon entering the matrix, the targeting sequence is cut off and the polypep is folded properly

1) Polypeptide transported through outer pore and starts going through the inner - STOP sequence in the polypepcauses it to leave the inner membrane pore and remain in the membrane. 2) Polypeptide gets completely into the matrix -A mitochon. protein called OXA 1 takes it and inserts it into the inner mitochondrial membrane 3) Multipassproteins are inserted via other Tim proteins

-Two pathways �In both cases, the polypeptide contains an intermembrane- space targeting signal 1) Polypeptide is initially inserted into the inner membrane. It is then CLEAVED �the piece remaining stays in the space 2) Direct delivery into the space

Getting into the outer membrane -Stop transfer sequences keep it on the outer membrane

Peroxisomalpolypeptides are made on free ribosomesand they are transported into peroxisomespost-translationally(like mitochondrial proteins)

These proteins contain a Peroxisomal Targeting Sequence (called PTS1)�usually only 3-4 a.a. long -A cytosolicprotein called Pex5 binds to PTS1

-A cytosolic protein called Pex5 binds to PTS1 -Pex5 also binds to a protein called Pex14, which is located on the peroxisomal membrane

PTS1 containing proteins are then translocated into the peroxisomelumen through a channel complex -PTS1 is NOT cleaved off in the process � Peroxisomalproteins can actually be pre-folded before being translocated(different from ER and mitochondria)

-the specific targeting signal is called NLS(nuclear localization signal); is a short sequence that has 5-10 amino acids - proteins called importins bind to it - transport proteins containing them through the nuclear pore into the nucleus

A protein called Ran(complexedto GTP) causes import into come off of the NLS-containing protein -Ran-GTP then ships importin back into the cytoplasm -In the cytoplasm, GTP is hyrdolyzedinto GDP --this causes Ran to come off of importin(importin is now free to bring more proteins into the nucleus)

Proteins can also contain signals called nuclear export signals(NESs) -These are recognized by exportins, which act in a similar way to importins

The general pathway by which proteins are transported From the ER to these other organelles(within vesicles) is called the secretory pathway. --Proteins can also move in the opposite direction (from the PM bac

All protein movement occurs via vesicles (very few instances where free proteins move from organelle-to-organelle) -What is the benefit of using vesicles? Protection, Speed, & Efficiency.

1) Proteins are packaged into a vesicle in one organelle 2) The vesicle is shipped to a second organelle 3) The vesicle fuses with that membrane, delivering the protein cargo (e.g. releasing it into the lumen) Note: Topology is maintained in different organelles! -Lumen of ER = Lumen of Golgi = Outside of cell

� Steps of the secretorypathway(cisternal maturation) a) Proteins made in the rough ER as described before -ER-resident proteins contain signals that either prevent them from moving on or actively bring them back each time they escape in a vesicle -KDEL & KKXX motifs. b) Proteins are then packaged into transport vesicles and shipped away from the ER -Many of these vesicles will Fuse together to form an intermediate compartment called the cis-Golgi network (CGN) -Again, ER-resident proteins escaping to the CGN are shipped back to the ER via vesicles -Very common c) This individual CGN cisternae(containing its cargo proteins) will then begin to movae towards the plasma membrane. d) Vesicles containing enzymes that reside in the cis region of the Golgi apparatus come in and with the moving cisternae -The cargo proteins are modified by these cis enzymes -This causes the cisternae to become part of the cisGolgi apparatus

e) The cisternae continues to move �vesicles containing enzymes that reside in the medial Golgi apparatus fuse with it (they also modify the cargo) -The cisternaenow contains cisGolgi enzymes and medial Golgi enzymes -The cis Golgi enzymes are finished doing their job and they're past where they want to be -They will be collected into a vesicle and shipped out of the cisternae (and go back and fuse with a new incoming cisternae) -The cisternae(w/ medialGolgi enzymes) is now part of the medial golgi f) New vesicles containing Trans-Golgi enzymes fuse with the cisternae -Those enzymes also modify the cargo proteins -Medial specific enzymes are packaged into a vesicle and sent out of the cisternae (these vesicles go back and fuse with a new incoming cisternae) -The resulting cisternaeis now part of the trans face of the Golgi apparatus g) Once the trans-Golgi specific enzymes are finished processing the cargo proteins, they too are packaged into vesicles and shipped out of h) The cisternaecontinues to move and eventually becomes part of another intermediate compartment called the Trans Golgi Network (TGN) i) The TGN is the organized branch point of the secretorypathway -Some vesicles emerge from TGN and are shipped to Lysosomes (delivering cargo proteins to that site) -Other vesicles go from TGN to the Plasma membrane (secreted proteins) -If those vesicles contain lumenalcargo proteins, the contents will be released from the cell following fusion with the plasma membrane

-Donor membrane (e.g. ER) contains integral membrane cargo proteins or lumenalcargo proteins bound to an integral membrane receptor -The cytosolic portions of these integral proteins serve as binding sites for cytosolic proteins that aggregate into a protein coat -Formation of this coat causes the membrane to bulb out into a vesicle ?Thus, the coat forms the vesicle and selects what gets in

1) COPII�forms coats on vesicles in the rough ER and directs them to the Golgi (ER --> Golgi) 2) COPI�forms coats in the Golgi and directs vesicles back (retrograde) to a previous Golgi compartment or to the ER (GOLGI ?GOLGI, GOLGI?ER) 3) Calthrin�forms on plasma membrane and TGN -Directs vesicles from TGN or the PM to a compartment called a late endosome(precursor to lyso) 4) ??�Forms on TGN and sends vesicles directly to the PM

-Proteins that use GTP degradation as an on-off switch (called GTPases)regulate coat assembly/disassembly -COPII uses a GTPase called SAR 1 -COPI and clathrinuse a GTPase called ARF -Sar1 and ARF tell the coat when its okay to form and when it should come off the free vesicle

Integral cargo proteins and receptors of lumenal cargo proteins contain various sequence motifs that are recognized by COPI, COPII, or clathrin (indirectly)

1. KDEL and KKXXsequences are recognized by COPI -Any proteins containing these motifs will attract COPI, which will initiate vesicle formation and target the vesicle to the ER 2. DXE Motif �recognized by COPII -These proteins will be shipped from the ER to the Golgi 3. YXXL & LL motif �recognized by clathrin(INDIRECTLY �more later) -Involved in TGN?endosomeor PM ?endosome

The binding of vesicle to target is mediated by several protein-protein interactions 1) v-SNAREs + t-SNAREs�Vesicles contain proteins on the surface called v-SNAREs. Target membranes contain surface proteins called t-SNAREs -v-and t-SNARES interact tightly with one another 2) Rabs+ Rab-effectors�Surface of vesicles contain proteins called Rabs, target membranes contain receptors for these Rabs called Rabeffectors (binding is regulated by GTP hydrolysis)

1. Interaction between a specific Rab(on the vesicle) and specific Rab-effector(on the target) allows the vesicle to dock onto the target 2. This docking gives the SNAREs time to interact -SNARE interaction pulls the vesicle very close to the target membrane -SNARE proteins have coiled-coil motifs (allows tight interactions to take place) 3. The vesicle membrane fuses with the target membrane (no idea how!) 4. Another protein called NSF comes in and actively unwinds the different SNARE proteins -This requires ATP hydrolysis

-unwinds SNARE complex

� Clathrinforms coats around vesicles in the TGN -These vesicles will be headed toward an intermediate compartment called the late endosome (which is a precursor to lysosomes) -Where do they go from the late endosome? � Clathrindoesn't bind to cargo proteins directly the way that COPI and II do -A group of proteins called adapter proteins (APs) bind to the cargo. Clathrinbinds to APs -AP1, 2, and 3 have been identified � located in different places (TGN, PM, lyso) � Each clathrinprotein consists of a heavy chain and a light chain -Three of these usually come together (interact) to form a three-legged structure called a triskelion

� Clathrin-coated vesicles pinch off from the donor membrane with the help of a protein called dynamin -Dynaminforms a noose-like structure around the vesicle "stalk" and squeezes it until the 2 sides meet -Dynaminis not involved in pinching of COPI/II vesicles -No one knows how they pinch off � Once the vesicle is off, the clathrin/AP coat comes off the vesicle -It will then fuse with the target membrane as described earlier (e.g. SNAREs)

Clathrinis also involved in forming vesicles at the plasma membrane when substances are brought into the cell via receptor-mediated endocytosis � Receptor-mediated endocytosis -Process that allows the cell to bring in a specific ligandfrom the outside environment 1) Receptor binds to ligandon the cell surface 2) That binding triggers the formation of a AP/Calthon coat on the underside of the PM (at that site) -This pulls the receptor-ligandcomplex into a vesicle 3) The vesicle (+ cargo) loses the coat and fuses with the late endosome -Late endosomes= Enlarged vesicles that have a low pH 4) The low pH of the late endosomecauses the ligandto come off of the receptor 5) That protein can be shipped to the lysosome(for degradation), to the TGN, or back to the surface

1) Add a radioactive amino acid to the cells for a very brief period of time (Pulse) -All new proteins made in that time will be radioactive 2) Add back the "COld" version of the amino acid 3) A labeled population of proteins will initially be in the rough ER and will begin to move through the secretorypathway 4) At various times after the end of the pulse, see where the radioactivity is -How do we detect the radioactive proteins in different organelles? a) Can Purify the different organelles and count how much radioactivity is in each (really only can separate ER from Golgi from PM �on a good day) b) Look where the radioactivity is by EM (old school) c) Monitor how the labeled protein is modified -Proteins in the ER will belargely unmodified -At each stage in the Golgi,proteins get different modifications

� Monitoring protein movement using fluorescence (more common now) 1) Label a specific protein population with a fluorescent dye (via an antibody or using GFP) 2) Take away the label 3) Using a microscope, watch how the protein moves through the cell over time (Can follow proteins coming into the cell or leaving the cell)

• Most families of viruses use the endocytic pathway as a means of gaining entry into the cell
• 1) Virus binds to a receptor on the cell surface, which triggers clathrin coat formation
• -The cell thinks the receptor is binding to a good ligand
• 2) Virus is brought into a vesicle and shipped to the late endosome
• 3) The low pH environment in the endosome helps trigger a change in the virus surface protein
• -This causes the virus membrane and the cell membrane to get closer
• 4) The 2 membranes fuse, releasing the virus into the cytoplasm ?Examples: Influenza and rhinoviruses � Many types of viruses use the secretory pathway as a means of getting newly assembled viruses out of the cell -Examples: Herpesviruses, smallpox, SARS, hepatitis B virus � Viruses "bud" into a ER or Golgi membrane, acquiring their outer envelope -The enveloped viruses (inside of a vesicle) hitch a ride and go fromthrough the secretory pathway until reaching the PM -Fusion of the vesicle with the PM releases the virus into the outside environment (the virus is the vesicle cargo) � These viruses can be shut down with drugs that inhibit the secretory pathway -Too toxic (ours cells need this pathway)