Ch 15: The system of Internal Membranes (Secretion)

ER-Golgi Intermediate Complex (ERGIC)

(protein sorting) system of membrane stacks located b/w the ER and the Golgi a checkpoint that ensures that proteins that should stay in the ER dont reach the golgi complex b/c traffic from the ER to golgi is non-selective (bulk flow)

Constitutive secretion

-operates in all cells continuously -unregulated secretion (bulk flow) -includes secreted proteins, membrane proteins and lipids, extracellular matrix proteins

Regulated secretion

-operates in some specialized cells (secretory cells) -these cell types release their secreted products only when stimulated by an external signal ex. endocrine, pancreatic, neurons

NLS (nuclear localization signal)

1 - 2 clusters of 4-5 positively larged resides (Lys, Arg) which are recognized by importin-α, an adaptor that connects the cargo to importin-β, the main transport mediator - not cleaved off - allows protein to be actively transported - 7 AA stretch, typically basic and charged AA in the middle of the polypeptide

Which statement about secretion is true? A) the membrane of a secretory vesicle will fuse with the plasma membrane when it discharges its contents to the cell's exterior. B) Vesicles for regulated exocystosis will not bud off the trans Golfi network until the appropriate signal has been received from the cell C) The signal sequence of proteins destined for constitutive exocytosis ensure their packaging into the correct vesicles. D) Proteins destined for constitutive exocytosis accumulate in the trans Golgi network until a signal is received.

A

nuclear envelope

A double membrane that surrounds the nucleus in the cell

All of the proteins below should be retained by the ER instead of being sent to the Golgi, except: A) SRP receptor B) SRP C) translocation channel D) signal peptidase E) hormone receptor

B) never entered ER E) needs to be sent to plasma membrane

All are examples of proteins that are transported into the nucleus through nuclear pores except: A) DNA and RNA polymerases B) transcription factors (TFs) C) chromatin assembly proteins (histones) D) ATP synthase E) lamins

D

Your research studies Fuzzy, a soluble protein that functions within the ER lumen. Given that info, which of the following statements must be true? A) Fuzzy has a C-terminal sequence that binds to SRP B) Only one ribosome can be bound to the mRNA encoding Fuzzy during translation C) Fuzzy must contain a hydrophobic stop-transfer sequence. D) Once the signal sequence from Fuzzy has been cleaved, the signal peptide remains in the ER membrane while Fuzzy is released.

D

What provides the energy for nuclear import?

GTP hydrolysis in the cytoplasm

During mitosis, what happens to the nuclear envelope?

It breaks down into small vesicles when nuclear lamina filaments are phosphorylated

Your friend works in a biotechnology company and has discovered a drug that blocks the ability of Ran to exchange GDP for GTP. What is the most likely effect of this drug on nuclear transport?

Nuclear transport receptors would be unable to release their cargo in the nucleus

What does the translocation cycle require?

Ran (GTP-binding protein) 25kDa

Protein import thru a nuclear pore step 3

Ran-GTP binds to the nuclear transport receptor, causing dissociation and release of the cargo protein in the nucleus

What drives the nuclear import cycle?

Ran-GTP diffusion

Function of Ran in active nuclear import

Ran-GTP is more abundant inside nucleus, therefore it will diffuse out into cytoplasm and then GTP is hydrolyzed to GDP by RanGAP creating a GTP gradient across the nuclear membrane. Ran-GDP diffuses back into nucleus, the GDP on the Ran protein is exchanged for GTP (catalyzed by RanGEF) in the nucleus

Protein import thru a nuclear pore step 5

RanGAP removes a Pi fro the GTP, yielding Ran-GDP in the cytoplasm

perinuclear space

The cavity between the two membranes of the nuclear envelope.

Nuclear lamina

a network of protein fibers that lines the inner face of the inner nuclear membrane, may have a role in gene regulation by regulating condensation patterns

Protein import thru a nuclear pore step 1

a protein complex forms the prospective nuclear protein with an NLS ( part of cargo protein) a nuclear transport receptor protein (importin) that recognizes and binds the NLS in the cargo protein

lamins

belong to a category of cytoskeletal proteins called intermediate filaments

2 types of transport from the golgi

constitutive secretion and regulated secretion

function of the golgi apparatus (complex)

further oligosaccharide modification of proteins + lipid sorting and packaging either for secretion or delivery to other parts of the endomembrane system

When does translation occur in a free ribosome?

if the synthesized protein is destined for the nucleus, chloroplast, mitochondrial or to stay in the cytosol

What does the N-terminal do?

initiates translocation

SV40 T-antigen

initiates viral DNA replication

GDP/GTP Exchange

inside the nucleus catalyzed by RanGEF Ran-GDP --> loss of GDP --> gain of GTP --> Ran-GTP --> binds importin + back to cytoplasm down the GTP gradient

Final destination of proteins synthesized on rough ER

lysosome, plasma membrane or out of the cell thru secretory vesicles

I Kappa B (IKB)

masks the NLS of the NF-KB, if phosphorylated and destroyed it releases NF-KB which is then bound by the nuclear transport receptor and translocated to the nucleus, where it can activate transcription required for immune response

SRP + SRP receptors

molecular match makers

GTP hydrolysis

occurs in cytoplasm and is catalyzed by RanGAP Ran-GTP is hydrolyzed --> loss of Pi --> Ran-GDP --> releases importin + back to nucleus down the GDP gradient

Nuclear pore

protein complexes that facilitate transport of molecules thru the nuclear pore

Endoplasmic Reticulum (ER)

protein processing

Golgi apparatus

protein sorting protein-containing vesicles from the ER enter the Golgi at the cis Golgi network proteins are then moved thru the medial stacks and released in vesicles from the trans golgi network

Translation

protein synthesis by ribosomes in the cytoplasm

SRP (signal recognition particle)

recognizes the signal sequence in the growing polypeptide, docks on the SRP receptor on the ER membrane, SRP is released as the growing polypeptide is passed to a translocation channel, translocation channel inserts the polypeptide into the membrane and transfers it across the lipid bilayer, signal peptidase removes the signal sequence from the new polypeptide

Active transport

requires energy, larger proteins and rRNAs, proteins contain a NLS (nuclear localization signal

KDEL

retention signal that ER resident proteins contain (Lys-Asp-Glu-Leu)

pancreatic cells

secrete exocrine digestive enzymes

endocrine cells

secrete hormones

neurons

secrete neurotransmitters

Signal sequence

secreted protein contain this at their amino terminus (first 8 hydrophobic AA), necessary for translocation into the ER, and after translocation it is removed binds to SRP and binds to translocation channel

What pathway do proteins synthesized on the rough ER take?

secretion pathway

Smooth ER

site of lipid synthesis and processing, constructs membranes and membrane systems for the cell, contains no ribosomes

Rough ER

site of protein synthesis and processing, disulfide bond formation, addition of cofactors, addition of oligosaccharides to lipids and proteins, has ribosomes

Passive diffusion

small molecules, small proteins <50kDa, no energy required

synaptic signaling

specialized type of regulated secretion - a nerve cell releases neurotransmitter molecules into a synapse, stimulating the target cell

What does the nuclear lamina provide?

structural support for the nucleus and attachment sites for the chromatin

Which way is the C-terminus of the transmembrane proteins always facing?

the cytosol

What is the outer membrane of the nuclear envelope continuous with?

the endoplasmic reticulum

Protein import thru a nuclear pore step 2

the entire protein complex (nuclear transport receptor plus the cargo protein) is translocated thru the nuclear pore by sequential interactions with nuclear pore proteins

Protein import thru a nuclear pore step 6

the nuclear transport receptor dissociates from Ran-GDP and is available to bind more cargo

Protein import thru a nuclear pore step 4

the nuclear transport receptor/Ran-GTP complex returns to the cytoplasm

What happens to the secreted products?

they are sorted into larger secretory vesicles that remain in the cytosol until a signal is received

NF-Kappa B

transcription factor that exists dormant in the cytoplasm in a complex with I Kappa B ex. human B cells

Pho4

transcription factor that exists in the cytoplasm phosphorylated (inactive) state ex. yeast cells the phosphate group blocks its NLS, Pho4 can be dephopshorylated which binds to the nuclear transport receptor to enter the nucleus and activate genes that regulate cell cycle

What happens if the synthesized protein is destined for the ER and Golgi as a transmembrane, lysosomal or secreted product?

translation occurs in ribosomes that are associated with the rough ER and the newly synthesized protein is translocated into the lumen (interior) of the ER