Folding, Assembly and Post-translational modification of Proteins

During insertion, nascent membrane proteins have to:

• adopt the correct orientation in the lipid bilayer
• undergo covalent modifications
• cleavage of the signal sequence
• N-linked glycosylation
• fold properly
• adopt their native state
• through interaction with ER-resident proteins such as chaperones

It is important to know that many of the proteins present in the lumen of the ER are in transit to other destinations. Others who are residents of the ER carry an "ER retention signal" composed of four amino acids at the C-terminus.

Two ER resident proteins are important:

1. protein disulfide isomerase (PDI) - catalyses the formation of disulfide bonds (S--S) from free sulfhydryl groups (SH) on cysteines
2. binding protein (BiP) - an hsp70-like chaperone that helps in translocation of proteins and also recognizes incorrectly folded proteins

Protein Translocation into ER + Signal Recognition Particle

The Endoplasmic Reticulum membrane is a busy place like an airport. Most of the integral membrane proteins and soluble proteins are co-translationally targeted, inserted and assembled here at sites referred to as translocons which consist of a group of membrane proteins that act in concert with ribosomes and molecular chaperones to facilitate:

• the insertion of integral membrane proteins into the lipid bilayer
• the translocation of soluble proteins into the ER lumen

ER Signal Sequences

Protein targeting to the ER membrane can occur co-translationally or post-translationally depending on the hydrophobicity and location of the signal sequence (SS) which consist of:

• a short sequence of hydrophobic residues
• a charged, positive N-terminal region on one side
• an uncharged, polar C-terminal region on the other side

In the post-translational process, the synthesized proteins are targeted and inserted (or translocated) after translation by the cytosolic ribosomes.

In the co-translational process, targeting of soluble and integral membrane proteins is mediated by the conserved Signal Recognition Particle.

The Endoplasmic Reticulum

The Endoplasmic Reticulum (ER) is a strikingly complex structure within the cell and has three morphologically distinct regions: 

1. Sheets of the nuclear envelope
2. A network of interconnected peripheral ER tubules
3. Peripheral ER sheets

Structure of ER, EMBO reports (2010) 11, 515–521

 All three regions exist within the continuous membrane bilayer and corelate with specialized ER functions. They can be easily detected by flourescence microscopy.

Protein Transport in Chloroplasts

Like mitochondria, chloroplasts must correctly localize proteins encoded in the nucleus and subsequently synthesized in the cytosol. Protein transport in chloroplasts is similar to that in mitochondria with a few exceptions:

Features	Mitochodria	Chroloplasts
Process occurs	post-translationally	post-translationally
Translocation complexes	separate in each membrane	separate in each membrane
Requires energy?	Yes	Yes
Requires signal sequences?	N-terminal, removed after import	N-terminal, removed after import
Requires chaperones?	Yes	Yes
Requires gradient across membrane?	Electrochemical (H+) in inner membrane	Electrochemical (H+) in thylakoid membrane
Requires ATP/GTP hydrolysis?	No	Yes

Mitochondrial Protein Transport

Despite the presence of the mitochondrial genome in the cell, only few mitochondrial proteins are encoded by the mitochondria itself. The vast majority are encoded in the nucleus and consequently synthesized (transcribed) in the cytosol as precursor proteins containing mitochondrial targeting elements that target them to the mitochondria.

Mitochondrial targeting elements exist as single or multiple units scattered along the length of the precursor and vary in terms of sequence, structure and location. 

The most common or "classical" presequence is an N-terminal stretch of 15–55 amino acids which is cleaved upon import.

It is assumed that mitochondrial protein import primarily occurs post-translationally. This necessitates the requirement for cytosolic chaperones to maintain the precursor proteins in their unfolded and import-ready states.

The mitochondria contain four compartments:

• outer mitochondrial membrane (OMM)
• intermembrane space (IMS)
• inner mitochondrial membrane (IMM)
• matrix

How do the cargo precursor proteins overcome the barriers posed by the mitochondrial membranes - especially the outer mitochondrial membrane?

Transport through the Nuclear Pore Complex

As described in the earlier post, the Nuclear Pore Complex (NPC) serves two key purposes:

• to form a barrier of selective permeability within the pore (preventing the passage of nonspecific macromolecules and at the same time allowing the free diffusion of water molecules, sugars and ions) and
• to facilitate transport of selected macromolecules across it

Molecules that go into the nucleus include inner nuclear membrane proteins as well as all the proteins found in nucleoplasm. On the other hand, proteins that go out of the nucleus include those associated with RNA (assembled into ribosomal subunits) and ribonucleoproteins (mRNPs).

Once transported, the NPC must also ensure that the molecules are retained in their respective cytoplasmic and nuclear compartments. This calls for regulation of nuclear transport at multiple stages.

The Nuclear Pore Complex

DNA is enclosed by the nuclear envelope consisting of two concentric membranes - outer and inner nuclear membranes which contain numerous protein complexes called the Nuclear Pore Complexes or NPCs.

The outer nuclear membrane is studded with ribosomes which synthesize proteins that are imported into the perinuclear space (the space between outer and inner nuclear membranes).

The Nuclear Envelope is perforated with Nuclear Pore Complexes

It is estimated that there are around 3000-4000 NPCs in a typical mammalian cell weighing about 125 megadaltons.

Protein Sorting

Protein Mobilization between compartments

Almost all proteins are synthesized on ribosomes in the cytosol but a few find their origin on the ribosomes in the mitochondria or chloroplasts.

These newly synthesized proteins carry out important functions in different organelles where they must be promptly delivered.

But how does the cell know which newly synthesized protein goes to what organelle?

Membrane-Bound Organelles

Since thousands of chemical reactions occur at any given time within a cell, it is imperative for the cell to segregate the chemical reactions so as to avoid "chemical chaos" from taking place inside the cell.

The cell, therefore, has evolved 2 strategies to overcome this problem:

Membrane Transport

• Cell membranes are permeable to large uncharged polar molecules (amino acids, glucose and nucleotides) and charged molecules (ions). Transfer of these water-soluble molecules depends on membrane transport proteins
•  If a cell is not to be torn apart by electrical forces, then the quantity of positive charge inside or outside the cell must be balanced by an almost exactly equal quantity of negative charge (outside or inside respectively)
• Nature has therefore employed mechanisms by which small molecules cross lipid bilayers
• passive diffusion → a molecule simply dissolves in the lipid bilayer, diffuses across it, and then dissolves in the aqueous solution at the other side of the membrane
• gases (O₂, CO₂), hydrophobic molecules (benzene) and small polar uncharged molecules (H₂O, ethanol) are able to dissolve in the lipid bilayer
• no membrane proteins are involved
• no external source of energy is required
• the net flow of molecules is always down their concentration gradient →  the direction of transport is determined only by the relative concentrations of the molecule inside and outside of the cell

The Cell Membrane

Cell Membrane

• All cells are enclosed by a thin, film-like membrane called the plasmalemma or more popularly as the plasma membrane
• Danielli and Davson (1935) proposed a “trilaminar model” according to which, the plasma membrane is formed of a bimolecular layer of phospholipids (35 Å thick) sandwitched between two layers of proteins (each 20 Å thick). The model was proposed even before the plasma membrane was seen under the electron microscope
• J.D. Robertson (1959) proposed a “unit membrane concept” according to which, all biological membranes shared the same basic structure:
• thickness of about 75 Å
• a characteristic trilaminar appearance when viewed with electron microscope
• the three layers are a result of the same arrangement of proteins and lipids as proposed by Danielli and Davson
• S. J. Singer and G. Nicolson (1972) put forward the “fluid mosaic model” of membrane structure which is presently the most widely accepted model.

Components of the Plasma Membrane

The Plasma membrane structure

• According to the fluid mosaic model, the cell membrane consists of a highly viscous fluid matrix of two layers of phospholipid molecules which serve as a relatively impermeable barrier to the passage of most water soluble molecules
• The plasma membrane contains lipids (32%), proteins (42%), carbohydrates (6%) and water (20%) although variations are always there
• Protein molecules or their complexes occur in the membrane, but not in continuous layer; instead, these occur as separate particles asymmetrically arranged in a mosaic pattern
  

Fundamental Processes - Overview of the Cell Cycle

Control of Cell Division

• Most cells have two major phases: mitosis and interphase often together referred to as the cell cycle
• For most tissues at any given time, only a few cells are in mitosis while the rest remain in interphase which is the period between divisions of the cytoplasm and is where a typical eukaryotic cell spends most of its life
• Some cells lose the capacity to divide altogether and stay in interphase indefinitely (for example in humans: nerve cells and muscle cells), while some divide regularly and others only occasionally

The Cell Cycle

Interphase consists of three sub-phases:

• G₁ is Gap 1 → the period just after mitosis and before the beginning of DNA synthesis
• S (synthesis) → which is the time when the cell’s DNA is replicated
• G₂ is Gap 2 → the time after S and prior to mitosis

Mitosis and cytokinesis are referred to as M-phase:

• the G₁→S transition commits the cell to enter another cell cycle