05A Protein Import [ Intro to cytosol ]

This organelle contains a little more than half of the total cell volume

Cytosol

What are the components of the cytoplasm

Cytosol and Cytoplasmic organelles

Where protein synthesis and degradation occurs

Cytosol

True or false: Intermediary metabolism occurs in the cytosol ( catabolic and anabolic reaction s )

True

What do you call the sustem of organelles that have topologically similar membranes or have similar structures

The endomembrane system

What are included in the endomembrane system or organelles that are membrane bounded compartments, with distinct functions due to the specialized mix of protein content

Er

GA

Lyso

Endosomes

Peroxi

Mito and plastids

This organelle is where the synthesis and modification of lipids and proteins for distribution occur

Endoplasmic reticulum

This organelle is where modification, sorting, and packaging of proteins for delivery occur

Golgi Apparatus

This organelle is where intracellular degradation occurs

Lysosomes

Organelle that facilitates the sorting of endocytosed material

Endosomes:

Organelle that performs various oxidative reactions

Peroxisomes

What are the different Protein Transport Mechanisms [ And their directionality ]

a. through pores or Gated Transport [ Nuclear Pore Complexes ] - bidirectional

b. transport across membranes [ Transmembrane protein translocators ] - unidirectional

c. transport by vesicle - bidirectional

What organelles do transport via pores or gates transport

Nucleus via Nuclear Pore Complexes

What organelles do transport via transmembrane protein translocators

Chloroplast

Mitochondria

Peroxisomes

What organelles do transport via vesicles

Endoplasmic Reticulum

Golgi Apparatus

Lysosomes

Endosomes

Directionality of Protein Sorting

Unidirectional

What defines a protein’s fate in protein sorting

Amino acid sequences

General Requirements for Protein Transport

1. Recognition signals or Signal sequences

2. Energy

3. Receptors

4. Translocators

These are specific amino acid sequences that gives the address where the protein needs to go or direct proteins to different locations

recognition signals or signal sequences

general term and other terms for recognition signals or signal sequences

General Term: protein sorting signal sequences

Other term: signal peptide/signal sequence/targeting sequence

What are the two types of recognition signals or signal sequences

1. Terminally located [ amino or carboxyl end ]

2. Internally Located

What enzyme usually cleaves Terminally located signal peptides or recognition signals

signal peptidase - FOR TERMINALLY LOCATED OKAY
*internal located are not usually cleaved

For terminally located signal sequences, how long are the continuous stretch of amino acids at the amino end of the protein to be sorted

15-60 [ THIS IS FOR THE AMINO END OKAY ]

This kind of signal sequence may be made up of patches that fold into specific 3D arrangement

Internally Located

Once a terminally located signal sequences gets folded, describe the orientation of these specific sequences

once it is folded, the signal sequence is located at the surface of the protein, so that it can easily bind with the receptor

Once the internally locates signal sequence gets folded, what is formed

Signal patch gets formed [ hydrophobic regions ] that exhibits molecular complementarity with a receptor

signal sequence for nucleus, perozisome, return to the ER

-KKKRK-

-SKL-COO-

-KDEL-COO

This requirement for protein transport powers the transport from different sources [ and what are these different sources ]

Energy

1. ATP, GTP,

2. Proton Motive Force

Or can be a spontaneous reaction

these protein transport requirement have molecular complementarity with the signal sequences

Receptors

these protein transport requirement are for recognition by other proteins that guides the proteins to their destination. These are either located on the cytosol or on the organelle membrane

Receptors

surface amino acids of protein are recognized by receptors to have stable binding based on what

chemical properties (molecular complementarity)

True or False: Receptor binding Recognize classes of proteins rather than individual protein species

True

Fate of receptors after unloading the cargo

They are recycle or reused

These are proteins that facilitate the transport that are embedded in an organelle membrane [ what are the functions as well ]

Translocators

Functions: for protein binding [ it allows also the integration of the protein into the membrane itself] and transport assistance

These protein sorting requirement can be modeled using thermodynamics and kinetics

Translocators

True or false: Protein import can be co-translational or post-translational

True

For post-translational import, protein synthesis occurs where

Free ribosomes in the cytosol that would get imported inside organelles [ Nucleus, Mito, Plastid, Peroxi ]

For co-translational import, protein synthesis occurs where

proteins synthesis occurs by being docked on the Rough ER ( translation and transcription are coupled )

This can then remain in the ER or go to GA and gets transported to the other organelles

Requirements for Nuclear Import

1. Signal Sequences: NLS - Nuclear Localization Signals

2. Energy: Hydrolysis of GTP via GTPase RAN (abundant in the cytoplasm)

3. Translocators: NPC

4. Receptors: Nuclear Transport Receptors [ Importin ]

Delivery sites for the nucleus

1. nuclear envelope

2. nucleoplasm

3. nucleus

the signal sequences for Nucleus import or the NLS [ Nuclear localization sequences ] are rich in what amino acids

Lysine (K) and Arginine (R) which are + charged AA

What is the Receptor for the import of proteins inside the nucleus called

Nuclear Transport Receptor - Importin

Explain the study that confirmed the role of NLS in protein import in this immunofluorescence micrographs

The normal antigen/signal sequence which is rich in lysine was successfully imported into the nucleus (A). When lysine was replaced by threonine, the T-antigen stayed in the cytosol as represented by immunofluorescence

What is the requirement for nuclear Export

1. Signal Sequences: NES - nuclear export signals (NES)

2. Energy: Hydrolysis of GTP via GTPase RAN (abundant in the cytoplasm)

3. Translocators: NPC

4. Receptors: Nuclear Transport Receptors [ Exportin ]

the signal sequences for Nucleus Export or the NES or Nuclear export signals are rich in what amino acids

leucine [ The Classic NES ]

What are the different processes that happens inside the nucleus that it is important to have protein import into the nucleus

1. ribosomal unit assembly

2. protein transcription

3. proteins for replication

4. proteins for chromosome packing

These act as an importin and exportin for different cargos in the Simultaneous import and export of proteins and it s examples

Bidirectional Karyopherins [ Importin 13, Exportin 1, Exportin 5 ]

True or false: In the SIMULTANEOUS IMPORT AND EXPORT OF PROTEINS, Cleaving is ABSENT (why)

True, Cleaving is absent for the NLS and NES since Importin and Exportin utilizes these as binding sites

This is the largest protein complex in the cell, that is made up of polypeptide subunits lining a pore in the nuclear envelope that are for import and export in the nucleus

Nuclear pore complex (NPC)

These components serves as protection during the exchange of components between nucleus and cytoplasm, or simply during bidirectional traffic.

Nuclear pore complex (NPC)

How does bidirectional Traffic occur in the nucleus and cytosol

Nucleus needs proteins imported from the cytoplasm, while RNA transcripts from the nucleus are exported to the cytoplasm [ During exchange or products ]

serve as a gate to regulate or prevent entry of materials through the nuclear envelope

Nuclear pore complex (NPC)

What are the parts of the Nuclear pore complex (NPC)

a. transporter ( the actual pore )

b. spokes ( attached to the anchor protein )

c. anchor proteins ( anchors the entire NPC to the membrane of the nuclear pore )

d. ring subunits ( where fibers are attached )

e. fibers ( faces the cytosol ) and Basket fibers ( faces the nucleoplasm ) - directly interacts with the protein so that it can determine whether if not it can pass thorough

specific part of the Nuclear pore complex (NPC) that directly interacts with the protein so that it can determine whether if not it can pass thorough

fibers

These are nuclear transport receptor protein (karyopherin) for import that binds NLS

Importin

Explain the Nuclear import of proteins

1. Importin + NLS of a cargo protein = Importin-NLS containing Cargo Complex

2. Goes through the NPC via diffusion with FG-nucleoporins, entering the nucleoplasm

3. RAN-GTP + Importin Cargo Complex [ Causing conformational change ] = RAN-GTP-Importin + Cargo Protein

4. RAN-GTP-Importin Exits the nucleoplasm via NPC

   • in the NPC there are cytoplasmic filaments with GTPase Activating Protein

5. Cytosolic side GTP Hydrolysis: RAN-GTP-Importin + GAP or GTPase Activating Protein = RAN-GDP + Importin

   • importin gets ready for another round

   • Ran-GDP is returned to the nucleoplasm + GEF = Ran-GTP

Explain the Nuclear Export of proteins

1. Nucleoplasmic side: Exportin 1-NES containing Cargo protein-Ran GTP complex

2. diffusises to the NPC via transient interaction with FG nucleoporins

3. GTP Hydrolysis in the cytosolic side

   • Cargo protein gets released and remains in the cytosol

   • Exportin 1 - recycled in the nucleoplasm

   • Ran-GDP + GEF ( in the nucleoplasm) = Ran GTP

Explain the RAN-Independent Export of mRNAs

1. Heterodimeric NXF1/NXT1 complex binds to mRNA protein (nucleoplasmic side)

2. NPC via transient interaction with FG- nucleoporins

3. mRNA e- erodimer NXF1/NXT1 —RNA helicase Dbp5—> NXF1/NXT1 _ mRNA

4. NXF1/NXT1 is recycled back to the nucleus by RAN dependent process

Ran-GDP is converted back to Ran-GTP via

Ran- GEF or Ran Guanine Nucleotide Exchange Factor

meaning of mRNPs

mRNA prtotein complexes [ complex of heterodimeric NXF1/NXT1 and mRNA ]

What enzyme facilitates the hydrolysis of complex of heterodimeric NXF1/NXT1 and mRNA

RNA helicase Dbp5

What causes the hydrolysis of RAN GTP when being transported to the cytosolic side via NPC

GTPase activating protein located at the cytoplasmic filaments of the NPC

Requirements for Nucleolus Transport

1. signal sequences: NOS or nucleolar targeting sequence

2. Energy : Hydrolysis of GTP via GTPase

3. receptor

4. translocator: NPC

what are the amino acids comprising of the signal sequences: NOS or nucleolar targeting sequence for the transport in the nucleolus

basic amino acids (argenine and lysine rich)

True or false: Cleaving is also absent in NOS just like NLS and NES as they serve as binding sited allowing the entry and exit of the molecules

True

What are usually transported in the nucleolus

ribosomal proteins since transcription of rRNA occurs here, the rDNA LOCUS consist of different genes that codes for genes in the rna product

This is a process by which ribosomes are produced, and what proteins are responsible for this process

Ribosomal biogenesis

• Ribosomal proteins

• nucleolar substructure

Explain the process of Ribosomal biogenesis

i. Transcription of rRNA (small and large subunits) and RNA transcripts for the ribosomal proteins

ii. Translation of ribosomal proteins by cytosolic ribosomes

iii. Import of ribosomal proteins into nucleolus

iv. Cleavage and modification of rRNA (small and large subunits)

v. Assembly of small and large ribosomal subunits (in nucleus)

vi. Export of small and large ribosomal into cytosol and assembly

Requirements for Cytosolic Transport

1. Signal sequence: Various ( no specific bec. of varying materials being transported)

2. Energy: Hydrolysis of GTP via GTPase - nucleus to cytosol only and others are ATP, PMF or spontaneous - depending on the organelles

3. Translocator

4. Receptor

Describe teh cleaving conditions of cytosol

cleaving will depend on the protein to be transported

FATES of Cytosolic proteins

1. binding to the plasma membrane

2. covalent modification

3. rapid degradation if misfolded (undergoes ubiquitylation)

Where does protein covalent modification happen ?

In the cytosol

In protein covalent modification, what do you call the process of attaching the +CHOS

Protein glycosylation

In protein covalent modification, what is an example of a coenzyme you attach ?

Biotin

In protein covalent modification, what do you call the enzyme that attaches a methyl group to the protein

Methyl transferases

In protein covalent modification, what do you call the enzyme that attaches a phosphate group to the protein

Kinases ( reversible - regulating the activity of the enzyme )

What do you call the enzyme that attaches an acetyl group during protein covalent modification ?

Acetyl carboxylase and acetylase - reversible

These are proteins that are for degradation, carrying degredation signals. It degrades by carrying destabilizing amino acids

Degrons

Memorize the destabilizing and stabilizing amino acids

Okay

What enzymes cause direct transfer of ubiquitin to the substrate

E2 and E3

What happens during conjugation in ubiquitin proteosome pathway

E3-substrate complex binds to E2, ubiquitin is theN transferred from E3 to substrate, E2E3 then dettaches

Multiple rounds of ubiquinylation occur, with ubiquitin attaching to one of the Lysines

Polyubiquitylation

To what specific part of the substrate will ubiquitin bind to during polyubiquitylation

Lysine ( k48m for proteins targeted for degradation ) on the previous ubiquitin

What is the enzyme that does protein degradation after polyubiquitylation

26S proteosome

Steps during UBIQUITIN-PROTEOSOME PATHWAYS

1. Activation via E1

2. Transfer via E2

3. Ligation via E3

4. Conjugation

5. Polyubiquitylation

6. Deubiquitylation and degradation via 26s proteosome

A macromolecular complex with multi catalytic proteinase activity for the breakdown of ubiquitinated proteins

26s Proteosomes

Parts of the 26s proteosomes

2- 19s regulatory particle

1 - 20s proteosome [ with Alpha and beta sub units ]