MCB 701 Exam 5: FULL SET

Intracellular compartments

- locations in cells that are enclosed/separated/distinct from one another usually due to membranes

- 1/2 compartments (organelles)

1/2 cytosol

What regions in the cell are topologically equivalent/continuous to one another ?

- nucleus and cytosol

- ER, golgi, lysosomes & other vesicles

What cellular activities help proteins become functional proteins in cells?

- protein folding

- covalent modification

- proteolysis

- compartmentalization

Protein compartmentalization

- the process of protein synthesis in terms of where each part physically takes place

- proteins are delivered to the proper target/location due to compartmentalization

The majority of protein synthesis occurs in

the cytosol

The signal hypothesis

- concept that proteins contain the info to target them to the right location in its am ac sequence

- includes topogenic, targeting, or sorting signals

If proteins remain in the cytosol, it is typically due to

lack of a topogenic signal

How are protein signals decoded in order to properly sort proteins?

- macromolecular receptors

- target proteins to their correct intracellular membrane

Proteins can enter targeted membranes/organelles via

gated or transmembrane transport

Integral membrane proteins (Tmem proteins)

- proteins that integrate into the membranes or organelles

- signal-dependent process

Non-tmem proteins can be targeted to locations/organelles via

vesicular transport

Types of protein transport/targeting in the cell

- gated transport

- transmembrane transport

- vesicular transport

Gated transport

- any movement of proteins b/w the cytosol and the nucleus

- facilitated by nuclear pores

Transmembrane transport

- any movement of proteins from the cytosol to a topologically different compartment (not continuous w/each other)

- cytosol > mitochondira, ER, peroxisomes

Vesicular transport

- any movement of proteins via vesicles to/from topologically equivalent spaces

- lumen to lumen transport

- ER <> golgi <> endosomes & lysosomes <> vesicles <> cell exterior

Vesicular transport moves what types of proteins

- soluble proteins

- Tmem/integral proteins

Proteins destined for vesicular transport usually have a sorting signal located

somewhere in the N-term of the protein (15-60 am ac ling)

Gated transport is also known as

nuclear transport

Nucleus structure (in reference to gated/nuclear transport)

- Nuclear envelope (inner and outer membrane

- Nuclear pore complexes

The nuclear envelope is continuous with

the ER

Nuclear pore complexes

- span the nuclear envelope

- mediates nuclear/gated transport

- mediated selective bi-directional transport of macromolecules

NPCs are composed of

- nucleoporins

- ~30 proteins

Nucleoporins are usually composed of what am ac repeats

- FG repeats

- Phe-X-Phe-Gly

Why are FG repeats in nucleoporins/NPCs important?

- facilitate karyopherin-bound cargo through NPC

- cargo-carries move from one repeat to the next to get the cargo through the NPC

NPCs allow for passive transport of

small solutes and particles (smaller than 5nm)

Nuclear localization signals (NLS)

- sorting signals that locate cytosolic proteins to the nucleus

- can be found anywhere in the protein

- surface exposure needed for recognition

NLSs tend to have what common am ac residues?

N, Q, H, K, R

(Asp, Glu, His, Lys, Arg)

Karyopherins

- family of nuclear import receptors

- bind to NLS regions on proteins in the cytosol

- forms cargo-carrier complexes that allow for movement into the NPC

NESs are recognized by what proteins?

- exportins (export receptors)

- bind to NESs to get the protein out of the NPC

Nuclear export signals (NES)

- specific am ac sequences that allow proteins to leave the nucleus

Ran

- guanine NT binding protein

- responsible for the directionality of protein movement b/w the nucleus and cytosol

Ran is found in what two conformations/forms?

- Ran-GTP (in nucleus)

- Ran-GDP (in cytosol)

Ran gradients are controlled by what proteins?

- Ran-GAP (GTPase-activating protein; in cytosol)

- Ran-GEF (Guanine NT exchange factor; in nucleus; has affinity for chromatin)

Ran-GTP, which is found in the nucleus, allows for

dissociation of cargo-importin complexes in the nucleus

Overall, Ran-GTP bound to a cargo-importR does what?

- releases cargo into a space

- importR-Ran-GTP stays bound until next cargo needs to be loaded

Mitochondrial protein import (Tmem transport)

- involves transient targeting signals

- requires ATP binding

- requires the electrochem gradient across the inner membrane

Types of mitochondrial sorting signals

- pre-sequences @ am/N term

- internal signals

- basic residues include +am ac separated by uncharged am ac

Types of protein translocators in the mito membranes

- TOM complex

- SAM complex

- TIM 22, 23, complex

- OXA complex

Which mito translocators are located in the outer or inner mito membrane?

- TOM and SAM: OMM

- TIMs and OAX: IMM

How is ATP utilized by mitochondrial import?

- ATP hydrolysis removes HSPs from proteins as it is shuttled into the IMS

- ATP hydrolysis used to move proteins from IMS into the mito matrix

If a protein is moved into the IMS and is destined to be an integral OMM protein, what facilitates this process?

- chaperone proteins bind to protein to prevent folding

- SAM complex can bind to protein and weave it in and out of the membrane (due to certain signals)

Peroxisome protein transport

- largely determined by C-term SKL sequences

- ^^ peroxisomal import signals

The SKL sequence typically include what am ac residues?

- Ser-Lys-Leu sequences

- canonical PTS

- bind to the import receptor on the peroxisome

Proteins destined for the peroxisome are typically

- bound to an SKL (cis-dominant)

- folded/assembled before import

Peroxins

- peroxisomal protein import receptors

- bind to peroxisomal targeting sequences (PTSIRs) usually in the cytosol

Zellweger syndrome

- a peroxisomal biogenesis disorder

- causes peroxisomes to be 'empty'

- causes over-accumulation of LCFAs & other lipids

The rough ER (rER) is continuous with

- the nucleus

- the smooth ER

ER functions

- secretory protein biosynthesis

- lipid/lipoprotein biosynthesis

- xenobiotic detoxification

- Ca2+ sequestration

When cells are homogenized, the rER can become

- vesicularized (not folded like pancakes anymore)

- smooth and rough (more dense) vesicles separate when put in solution

Secretory proteins are made on/processed

- membrane bound ribosomes

- segregated into vesicles

- contain transient signal peptides

The ER targeting/translocation targeting pathways of proteins was established by

reconstitution of the process in a cell free system

Signal peptide structure

- typically at N-term

- about 13-30 am ac long

- position 1: small

- position 3: no aromatic ring, no charge, not polar

General translocation of soluble ER targeted protein

- protein is synthesized in the cytosol

- signal sequence is recognized by the ER receptor

- protein is moved into the ER

- signal peptidase in the ER cleaves the signal sequence; protein is now freely floating in the ER

signal recognition particle (SRP)

- protein complex that binds to the signal sequence on the protein

- binds to the ribosome and signal sequence as the protein is being synthesized

What complex moved ER destined proteins to the ER surface?

the SRP bound to the signal sequence + active ribosome

The SRP-signal sequence binds to what on the ER surface?

- SRP receptor protein

- protein binds to the signal sequence

- allows protein translocator to move the protein into the ER

SecY

- an ER translocator protein

- forms a channel structure

The SecY ER protein translocator can be found in what two states?

- closed: plug prevents proteins from entering ER

- open: allows signal peptide and growing polypeptide to move into the channel

The signal sequence is 'dual recognized' by both

- the protein translocator

- the signal peptidase

Types of protein translocation into the ER

- co-translational translocation

- post-translational translocation

Co-translational translocation

the protein is synthesized while it is simultaneously being moved into the ER

Post-translational translocation

the protein is completely synthesized before being brought into the ER

The integral parts of proteins (for insertion into a membrane) are alwasy

-hydrophobic

- allows it to sit in the hydrophobic regions of bilayers

- ~19-21 am acids

Integral membrane proteins are properly placed into membranes due to

- start transfer sequences

- stop-transfer sequences

Types of integral/Tmem proteins

- Type I

- Type II

Type I tmem proteins

- COO- term is outside (in the cytosol)

-NH3 term is on the inside (ER lumen)

For type I tmem/integral proteins, the start transfer sequence would be located

on the N term

For type I tmem/integral proteins, the stop transfer sequence would be located closer to the

C term of the protein

Type II tmem/integral proteins

- COO- term is on the inside (ER lumen)

- NH3+ term is on the outside (cytosol)

For type II tmem/integral proteins, where would the start and stop transfer sequences be located?

- start: C term

- stop: N term

For multipass tmem/integral proteins, you need either

- start/stop transfer sequences located towards the middle of the protein sequence

- multiple start/stop transfer sequeunces

What is an example of translocated protein modification?

- N-liked oligosaccharide modification

- adds oligosaccharides towards the N term of the protein

What are other methods/ways proteins can associate/attach to membranes?

- amide links

- thioester links

- thioether links

(all are considered anchors)

GPI-anchored proteins

- attaches proteins to GPI (lipid-linked membrane proteins)

- sorts proteins for apical sorting (cell surface)

- converts secretory proteins to cell surface proteins

GPI-anchored protein precursors are made with

- N term signal peptide

- 20-30 am ac C term that is hydrophobic

GPI-anchored proteins anchor into membranes due to

- C-term hydrophobic domain

- replaced by another GPI moiety which actually anchors the protein to the membrane

What is the major intersection b/w the biosynthetic-secretory ptw and the endocytic ptw?

- lysosomes

- endosomes are delivered to the lysosome

- proteins/secretory proteins are delivered to the lysosome

What are the major sites of macromolecular turnover in euk cells?

- lysosomes

- proteasomes

Lysosomes

- membrane bound organelles that contain digestive hydrolases

- works w/rapidly decreasing pH via ATP H+ pumps (vacuolar H+ ATPase

Lysosome shape determines

- different stages in lysosome maturation cycle

- late endosome > endolysosome > lysosome facilitated by fusion events

Overall, lysosomes are viewed as a

heterogenous collection of distinct organelles

Lysosomal membrane structure

- 7-10 nm thick bilayer

- high carb content

- contain lysosomal membrane proteins (LMPs/LAMPs)

Lysosomal membranes are highly glycosylated because

it helps protect the lysosome inner membrane from the acid hydrolases inside of the lysosomes

What are the common LMPs/LAMPs in lysosomes?

- LAMP 1/2

- LIMP 1/2

What are the general functions of lysosomal membranes?

- form mechanical borders to contain catabolic enzymes

- establishes pH gradients b/w lysosome & cytosol

- mediates fusion w/plasma membrane for exocytosis

- can selectively move metabolites into the cytosol

How are lysosomal hydrolases and LMPs often delivered to lysosomes?

- mannose-6-phosphate tags (m6p)

- a signal patch on the hydrolase signals the addition of m6p to lysosomal

What enzyme adds M6P to lysosomal hydrolases?

GlcNAc phosphotransferase

How does GlcNAc phosphotransferase adds M6P to lysosomal hydrolases?

- signal patch on lysosomal hydrolase binds to enzyme

- M6P binds to the enzyme

- M6P is then attached to the hydrolase and released

the addition of M6P to lysosomal hydrolases are added in what organelle?

- the golgi

- multiple M6Ps are added to the hydrolase to ensure it goes to the lysosome

Once M6P is attached to a lysosomal hydrolase, how does it reach the lysosome?

- vesicular transport

- M6P binds to the M6PR on the inside of the golgi

- a clathrin coat pinches off the vesicle, which moves to the lysosome

- the hydrolase is dropped off into the lysosome

How are M6P receptors recycled back to the golgi?

- the low pH of the lysosome allows the M6PR to dissociate

- M6PRs cluster in recycling endosomes that return to the golgi

What are alternative signal molecules used to target hydrolases to lysosomes?

- Limp2: utilized by B-glucocerebrosidase

- B-gluCB is a lysosomal membrane protein

Lysosomal Membrane Proteins (LMPs)

- proteins that are integral to the lysosomal membrane

- delivered/transported to the lysosome differently than hydrolases

What are the two main methods LMPs are delivered to lysosomes?

- normal/regular secretory ptw (move via endocytic vesicles; the default secretory ptw)

- direct lysosomal sorting (M6P independent, tyrosine/dileucine, clathrin dependent/independent)

What are three methods used to deliver macromolecules to lysosomes?

- phagocytosis

- endocytosis

- autophagy

Phagocytosis

- cellular engulfment of large particles (>250 nm)

- utilized by phagocytes for clearance of pathogens and debris

- signal induced process via surface receptors (polarizes actin filaments to bring particles into cell)

Types of endocytosis

- clathrin dependent (clathrin mediated; CME)

- caveolae dependent (caveolin mediated; CavME)

- nonclathrin/caveolae endocytosis

Phosphoinositides & metabolism (PIP2 and PIP3)

- regulates vesicular transport

- only certain signaling molecules can bind to certain PIs/PIPs

- usually located in membranes, which aid in signaling capacity

Example of PI/PIP involvement in phagocytosis

- PIP2 is utilized to form the initial outreach of the PM to start engulfing the cell

- PI3 Kinase converts PIP2 > PIP3

- PIP3 is utilized to form the complete phagosome/completely engulf the cell

Macropinocytosis

- bulk fluid/nutrient uptake by the cell

- signaled/triggered in most cells; active in DCs

- forms actin ruffles of the PM that extend outward to bring materials in

- recycled back to the PM or lysosome after cargo delivery