Lecture 8: Intracellular Compartments and Protein Sorting Flashcards by Allie Skinkle

3 features of cellular compartmentalization?

-segregation of metabolic function -organelle-specific environments -membrane surfaces

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Segregation of metabolic function

-glycolosis in cytoplasm -Krebs cycle in mitochondria

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Organelle-specific environments

-redox potential in ER -changing PH in ER -acidic PH of lysosomes -glycosylation reactions in the ER and golgi

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Membrane surfaces

-lipid synthesis -PH gradients and ATP production

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What are the three types of transport between compartments?

-gated transport -transmembrane transport -vesicular transport

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Identify the type of transport in red

gated transport

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Identify the type of transport in blue

Transmembrane transport

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Identify the type of transport in green

vesicular transport

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How does a protein “know” where to go?

Specific elements of a protein, mostly primary amino acid sequence, direct protein traffic

– Signal sequences

– Signal patches

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What signal sequence is this?

import into nucleus

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What signal sequence is this?

Return to ER

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What signal sequence is this?

Import into mitochondria

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What signal sequence is this?

Import into plasmid

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What signal sequence is this?

import into peroxisomes

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What signal sequence is this?

Import into ER

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What signal sequence is this?

Return to ER

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Gated transport occurs___

from the cytoplasm to the nucleus

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The nuclear envelope is directly connected to the ___

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Nuclear pore complex

>125 MegaDaltons

>30 proteins (nucleoporins)

~3,000-4,000 NPCs per nucleus

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• Molecules less than ____ Da freely diffuse through the NPC

5,000

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Protein between _____ Da diffuse through more slowly proportional to their size

20-60,000

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Proteins greater than ___ Da require help to enter

60,000

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How do “big” proteins enter and exit the nucleus?

• They use a regulated (gated) pathway that relies on specific import signals or nuclear localization signals

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• Nuclear localization signals (NLSs) bind to…

specific receptor proteins that direct import into the nucleus through the NPC

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The nuclear localization signal (NLS) was identified by systematically deleting or modifying sequences in \_\_\_\_\_

SV40 T-antigen

A change as small as ___ can disrupt the activity of an NLS

one amino acid

Functionality as an NLS is ___ its location within the polypeptide

independent of

\_\_\_ bind NLSs and nucleoporins

Nuclear import receptors

Nuclear import receptors are a family of related protein called ____ or \_\_\_\_

importins or karyopherins

These protein bind “cargo” via the NLS in the cytosol and then bind to the NPC

nuclear import receptors (importins)

• Specific nucleoporins form tentacle-like fibril composed of amino acid repeats \_\_\_

- FG (Phenylalanine-Glycine) - Importins bind to these FG repeats

Different families of import receptors recognize different \_\_\_\_

NLSs (nuclear localization signals)

Some cargos require an ___ that contains the NLS

adapter protein

• Nucleocytoplasmic transport is \_\_\_\_

bidirectional

• Large molecules like _____ must exit the nucleus and enter the cytoplasm through the NPC

new ribosomal subunits and RNA molecules

• Nuclear export is very similar to import, but topologically reversed....

- Depends on nuclear export signals (NES) - Depends on export receptors (exportins/ karyopherins)

Ran GTPase

A small G-protein that provides energy and directionality to nucleocytoplasmic transport

Like all small G-proteins, ___ is a molecular switch that changes activity based on a bound \_\_\_\_

Ran, nucleotide

• The gradient of the two conformational forms of Ran drive ____ in the appropriate direction

nuclear transport

The Ran GTPase cycle

How Ran provides directionality

RanGTP cargo displacement

Ran-GTP and cargo binding to an import receptor is...

mutually exclusive

The nuclear envelope disassembles during mitosis

The ER network composed of sheets and tubules is regulated by \_\_\_

fusion and fission

Posttranslational transmembrane transport

* Mitochondria * Chloroplasts * Peroxisomes

Mitochondrial function

* Energy (ATP) production by oxidative phosphorylation * Intermediary metabolism (TCA cycle) * Lipid metabolism – Catabolism (beta-oxidation) – Synthesis

While mitochondria contain DNA and protein synthesis machinery, the majority of mitochondrial proteins are _____ encoded genes that are postranslationally imported

nuclear

\_\_\_ and ____ regulate mitochondrial shape

Fusion and fission

Mitochondrial matrix targeting sequence

* 20-50 amino acids * Rich in hydrophobic, positively charged basic (R,K) and hydroxylated (S,T) amino acids * Tend to lack acidic residues (D,E) * Form amphipathic alphahelices * Cleaved off the mature protein by a matrix protease

Translocation into the mitochondrial matrix depends on \_\_\_\_

a signal sequence and protein translocators

In mitochondrial protein imports, precursor proteins are imported as \_\_\_\_

unfolded polypeptide chains

Proteins are imported into the matrix at ___ that join the inner and outer membrane

contact sites

\_\_\_ and ____ are used to drive import into mitochondria

ATP hydrolysis and a proton gradient

Repeated cycles of ATP hydrolysis by ___ complete the import process

mHSP70

Transport to the inner membrane and inner membrane space requires ___ signals

two

• Translocase of the Outer Membrane (TOM)

* Required for the import of all nuclear encoded genes * Helps insert membrane proteins into the outer membrane

Sorting and Assembly Machine (SAM) complex

– Helps insert and fold beta-barrel proteins

What does TIM stand for?

• Translocase of the Inner Membrane (TIM)

TIM23 complex

• Spans both membranes ## Footnote • Mediates translocation of proteins in to the matrix space and inner membrane proteins

TIM22 complex

Mediates the insertion of a subclass of inner membrane proteins

OXA complex

• Mediates the insertion of inner membrane proteins synthesized in the mitochondrial matrix

Protein translocators in the mitochondrial membrane

Which protein translocaters are in the outer mitochondrial membrane?

- TOM complex - SAM complex

Which protein translocaters are in the inner mitochondrial membrane?

- TIM22 complex - TIM23 complex - OXA complex

The negative membrane potential helps “attract” the\_\_\_\_\_ charged mitochindrial targeting signal

positively

The integration of Porins (Beta-barrel proteins) into the outer mitochondrial membrane is accomplished by what protein translocators?

TOM and SAM

Protein import into the mitochondrial matrix is accomplished by which two translocators?

TOM and TIM23

How are proteins targeted to the inner mitochondrial membrane?

TOM/TIM23 path but with a stop transfer sequence OR TOM/TIM23 path and OXA complex

How are proteins targeted to the inner membrane space of the mitochondria?

TOM/TIM23/OXA then protease cleaves the second signal sequence OR TOM/TIM23 and stop transfer sequence, protease cleaves stop transfer sequence

How do you target to the inner membrane space through the outer membrane?

TOM complex, then protein oxidized by Mia40

What are the two complexes responsible for protein import into the stroma of the chloroplast?

TIC (inner membrane) TOC (outer membrane)

What are the four ways to translocate into the thylakoid space?

Sec pathway SRP-like pathway TAT pathway spontaneous insertion

Peroxisomal Functions

• House oxidative enzymes – Oxidases produce peroxide (H202), hence the name peroxisome – Contains catalase to degrade H202 • Lipid metabolism – fatty acids beta oxidation * Exclusively peroxisomal in yeast and plants * Shared with mitochondria in animal

\_\_\_\_\_ do not contain DNA and all proteins are imported posttranslationally

peroxisomes

At least ___ peroxisomal targeting sequences (PTS) are known for matrix proteins

two

\_\_\_\_ import is fundamentally different from other organelles since fully-folded proteins can be imported

peroxisome

\_\_\_ is a specific protein that catalyzes protein import into peroxisomes

Pex19

\_\_\_ genes or \_\_\_, are proteins responsible for the generation and maintenance of peroxisomes

Pex genes or peroxins