Advanced Cell Biology good and best ppt on the mrna transport (1).pptx

Transport Across Biological
Membranes
By: Barnabas Tefera, Gashaw Yimer & Rafatuel Sija
Addis Ababa University, Institute of
Biotechnology –
Department of Molecular Biology

Contents of The Presentation
Protein Transport Across the Endoplasmic Reticulum
Rapoport TA. Protein transport across the endoplasmic reticulum membrane. FEBS J. 2008 Sep;275(18):4471-8.
Ratcheting mRNA Out of The Nucleus
Stewart M. Ratcheting mRNA out of the nucleus. Mol Cell. 2007 Feb 9;25(3):327-30.
Glucose Transport Across Plasma Membranes: Facilitated Diffusion Systems
Baldwin, S. A., & Lienhard, G. E. (1981). Glucose transport across plasma membranes: facilitated diffusion systems. Trends in
Biochemical Sciences, 6, 208-211.
Our Presentation Exclusively Focuses On Discussing These Research Titles
B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 2

Protein Transport Across the
Endoplasmic Reticulum
By Tom A. Rapoport

Introduction
Protein translocation across the eukaryotic endoplasmic reticulum (ER) membrane is a decisive
step in biosynthesis.
Proteins include soluble ones for secretion or ER lumen and membrane proteins for the plasma
membrane or other organelles.
Soluble proteins have cleavable N-terminal signal sequences with 7 to 12 hydrophobic amino
acids.
Integral membrane proteins have transmembrane (TM) segments with about 20 hydrophobic amino
acids.
Both protein types use a common machinery for transport: a protein-conducting channel.
The channel allows polypeptides to cross the membrane and permits lateral exit of hydrophobic
TM segments of membrane proteins into the lipid phase.

 In bacteria, translocation of secretory and membrane proteins occurs through a homologous channel in
the plasma membrane.
 The translocation channel is formed by an evolutionarily conserved heterotrimeric membrane protein
complex: Sec61 in eukaryotes and SecY in bacteria and archaea.
 The a-subunit of the complex forms the channel pore, demonstrated by systematic crosslinking
experiments.
 Reconstitution experiments show that the Sec61/SecY complex is the only essential membrane
component for protein translocation in mammals and bacteria.
 The channel has an aqueous interior, demonstrated by electrophysiology experiments and measurements
of fluorescence lifetime of probes in a translocating polypeptide chain.
Continued

Protein translocation across the eukaryotic endoplasmic reticulum (ER) membrane is a crucial step
in biosynthesis. Different proteins, including soluble and membrane proteins, undergo
translocation. The translocation process involves a protein-conducting channel.
Co-translational translocation is a common mode involving the ribosome as the major partner. Co-
translational translocation starts with a targeting phase, guided by the signal recognition particle
(SRP). GTP hydrolysis during translation provides energy for translocation.
Some proteins undergo post-translational translocation after completion.
Different Modes of Translocation

Figure 01 - Model of co-translational
translocation
The polypeptide chain moves from the
tunnel inside the ribosome into the
membrane channel. The energy for
translocation is provided by GTP
hydrolysis during translation.

In yeast and likely all eukaryotes, post-translational translocation involves a ratcheting mechanism
with the Sec62/63 membrane protein complex and the ER luminal protein BiP. BiP serves as a
Brownian ratchet, preventing backsliding of the polypeptide and allowing forward movement until
complete translocation.
In eubacterial post-translational translocation, polypeptides are pushed through the channel by the
cytosolic ATPase SecA. SecA has two nucleotide-binding domains, and the mechanism of how its
movements push the polypeptide through the channel is unclear.
Bacterial translocation requires an electrochemical gradient across the membrane. Archaea likely
have both co- and post-translational translocation, but the mechanism of post-translational
translocation is unknown due to the absence of SecA, Sec62/63 complex, and BiP.
Continued

Figure 02 - Model of post-translational
translocation in eukaryotes.
A Brownian ratcheting mechanism is responsible for
moving a polypeptide chain through the membrane.
Translocation might be mediated by oligomers of
the Sec61p complex, as in the other modes of
translocation.

The crystal structure of an archaeal SecY complex provides valuable insights into the function of
the translocation channel.
This structure is likely representative of all species, supported by sequence conservation and its
similarity to a lower resolution structure of the Escherichia coli SecY complex determined by
electron microscopy (EM) from 2D crystals.
Structure and Function of Translocation
Channel

1. **Composition**: The Archeal SecY Complex is composed of multiple protein subunits that
work together to facilitate the translocation of proteins across cellular membranes.
2. **Transmembrane Domains**: It contains transmembrane domains that span the lipid bilayer of
the membrane, providing a channel for proteins to move across.
3. **SecY Subunit**: The SecY subunit is a critical component of the complex that forms the central
channel through which proteins pass during translocation.
4. **SecA Interaction**: The Archeal SecY Complex interacts with a molecular motor protein
called SecA, which provides the energy required for protein translocation.
5. **SecE and SecG Subunits**: These subunits assist in stabilizing the complex and regulating the
translocation process.
6. **Conformational Changes**: The structure of the Archeal SecY Complex undergoes
conformational changes to allow the passage of proteins through the channel.
7. **Evolutionary Conservation**: Certain structural features of the Archeal SecY Complex are
evolutionarily conserved across different species, highlighting the importance of this complex in
cellular function.
Key Features of the Archeal SecY Complex Structure

Figure 03 - Structure of the
translocation channel
A) View from the cytosol of the X-ray structure of
the SecY complex from Methanococcusjannaschii.
The two halves of SecY are colored blue (TM 1–5)
and red (TM 6–10). The plug is shown in yellow
and pore ring residues are shown in green. The
purple arrow indicates how the lateral gate opens.
The black arrow indicates how the plug moves to
open the channel across the membrane. (B)
Cross-section of the channel from the side.

In the translocation process, several key points occur to facilitate the movement of proteins across
cellular membranes. Here are some important steps:
1. **Protein Targeting**: The protein destined for translocation is tagged with a signal sequence that
directs it to the translocation machinery on the membrane.
2. **Recognition and Binding**: The signal sequence is recognized by a receptor on the membrane,
leading to the binding of the protein to the translocation complex.
3. **Translocation Channel Formation**: A translocation channel is formed by components of the
translocation complex, such as SecY in bacteria, through which the protein will pass.
4. **GTP Hydrolysis**: Energy in the form of GTP hydrolysis is often required to power the
translocation process. Molecular motors like SecA in bacteria provide this energy.
Key points in the translocation process

5. **Protein Movement**: The protein is translocated through the channel in a stepwise
manner, involving both the movement of the protein and conformational changes in the
translocation complex.
6. **Signal Sequence Cleavage**: Once the protein is fully translocated, the signal
sequence is cleaved off, allowing the mature protein to fold correctly and function within the
cell.
Understanding these key points in the translocation process is crucial for grasping how
proteins are correctly transported to their intended cellular locations.

In the translocation of a polypeptide chain through the Sec61/SecY complex, although the pore is formed by a
single molecule, it appears to involve oligomers. This observation is supported by experimental findings:
1. **Defective SecY Rescue Experiment**: In experiments where a SecY molecule is defective in SecA-
mediated translocation, it can be rescued by covalently linking it with a wild-type SecY copy. This indicates
that the presence of multiple SecY molecules working together can compensate for deficiencies in individual
units, suggesting oligomeric involvement.
2. **Disulfide Bridge Crosslinking**: Disulfide bridge crosslinking experiments have shown that SecA interacts
with a nontranslocating SecY copy and moves the polypeptide chain through a neighboring SecY copy. This
interaction between SecA and different SecY copies implies a collaborative effort among multiple units in the
Oligomeric Translocation Channels

The Sec61/SecY complex likely forms oligomers during co-translational translocation.
Here's a breakdown of the process:
1. **Initial Binding and Conformational Changes**: - When a ribosome/nascent chain/SRP complex
binds to the SRP receptor, a conformational change in the SRP exposes a site on the ribosome where
a single Sec61/SecY molecule could bind.
2. **Proximity of Sec61/SecY Molecule**: - Recent EM structures suggest that a single Sec61/SecY
molecule is close to the point where a polypeptide exits the ribosome, serving as the translocating
copy initially.
3. **Association of Additional Sec61/SecY Complex**: - At a later stage of translocation, an additional
copy of the Sec61/SecY complex may associate, potentially stabilizing the ribosome-channel junction
and facilitating the recruitment of other components like signal peptidase and oligosaccharyl
transferase.
Key Points Regarding The Involvement of Oligomers in
Translocation

4. **Role of Dissociation**: - Dissociation of Sec61/SecY oligomers could aid in the release of the
ribosome from the membrane after translocation termination, indicating a dynamic process.
5. **Flexibility in Channel Partners**: - Dissociable oligomers could allow the Sec61/SecY
complex to change channel partners and modes of translocation, showcasing adaptability in the
6. **Clarification on EM Structures**: - While early EM structures suggested the presence of
multiple Sec61 molecules, recent evidence indicates the presence of only one Sec61 molecule, with
additional density likely attributed to lipid and/or detergent rather than multiple Sec61 units.

During the synthesis of a membrane protein, hydrophobic transmembrane (TM) segments
move from the aqueous interior of the translocation channel through the lateral gate into
the lipid phase of the membrane. Two proposed mechanisms for this process are:
1. Continuous Opening and Closing of the Lateral Gate:
The lateral gate of the translocation channel may continuously open and close. This movement
exposes polypeptide segments within the aqueous channel to the surrounding hydrophobic lipid
phase.
A potential variation involves a 'window' in the lateral gate, allowing lipid hydrocarbon chains to
contact the translocating polypeptide while preventing charged head groups from entering the
channel.
Polypeptide segments inside the channel would partition between the aqueous and hydrophobic
environments.
Support for this model comes from photo-crosslinking experiments and the correlation between a
hydrophobicity scale and a peptide's tendency to span the membrane.
Membrane Protein Integration

2. Alternating Movement of Hydrophilic Segments:
Hydrophilic segments between the TM segments may alternately move from the ribosome through the
aqueous channel to the external side of the membrane or emerge into the cytosol between the ribosome and
channel through a visualized 'gap' in EM structures.
Regarding the orientation of the first TM segment of a membrane protein:
The N-terminus of the first TM segment can be on either side of the membrane depending on the amino
acid sequence of the protein.
If the first TM is long and the preceding sequence is not retained in the cytosol due to positive charges or
folding, the N-terminus can flip across the channel and exit laterally into the lipid phase.
When the N-terminus is retained in the cytosol, and the polypeptide chain is further elongated, the C-
terminus can translocate across the channel, inserting the polypeptide as a loop, as observed in secretory
proteins.
Continued

The translocation channel plays a crucial role in preventing the free movement of small
molecules, such as ions or metabolites, across the membrane. The crystal structure suggests a
simple model for maintaining the membrane barrier:
1. Closed Resting Channel:
The resting state of the channel is closed, consistent with electrophysiology experiments
indicating impermeability to ions and water in the absence of other components.
This closed state serves as a barrier to prevent the passage of small molecules.
2. Active Channel with Pore Ring Seal:
In the active state, the pore ring fits around the translocating polypeptide chain like a gasket,
restricting the passage of small molecules.
During the synthesis of a multi-spanning membrane protein, the seal alternates between the
nascent chain in the pore and, once the chain has left the pore, by the plug returning to the center
of Sec61/SecY.
Maintaining The Permeability Barrier

3. Plug Deletion Mutants:
Surprisingly, plug deletion mutants in yeast and E. coli are viable with
moderate translocation defects. Crystal structures of these mutants show
that new plugs form from neighboring polypeptide segments.
These new plugs still seal the closed channel but lack many interactions
that normally keep the plug in the center of SecY. This leads to continuous
channel opening and closing, allowing translocation of polypeptides with
defective or missing signal sequences.
Poor conservation of plug sequences among Sec61/SecY channels
supports the idea that promiscuous segments can seal the channel, locking
it in its closed state.
Continued

Ratcheting mRNA Out of
The Nucleus
By Stewart M.

The export of mRNA is a complex process, distinct from other nuclear trafficking
pathways, as it requires completion of transcription, splicing, and processing before
mRNA is exported.
While unique features characterize mRNA nuclear export, it shares a common sequence
of steps with other nuclear trafficking pathways, such as nuclear protein import/export
and the export of smaller RNAs.
Introduction

Con.t
These pathways involve three key steps:
The generation of a cargo:carrier complex in the donor compartment,
Translocation of this complex through nuclear pore complexes (npcs), and
 The release of cargo in the target compartment,
Followed by the recycling of the carrier.

1. Cargo:Carrier Complex Formation:
Involves the generation of a complex in the donor compartment.
Common steps shared with other nuclear trafficking pathways.
2. Translocation Through NPCs:
Cargoes are transported through NPCs facilitated by carrier molecules.
Interactions with NPC proteins, specifically FG-nucleoporins with distinctive sequence
repeats, play a role in facilitating passage through NPCs.
3. Release of Cargo and Recycling of Carrier:
In the target compartment, the cargo is released, and the carrier undergoes recycling.
Key Points About nuclear trafficking pathways,
Including mRNA Export

4. Distinctive Features of mRNA Export:
mRNA export primarily employs the Mex67:Mtr2 heterodimer (TAP:p15 or
NXF1:NXT1 in metazoans) as carriers, distinct from the b-karyopherin
superfamily commonly used in other nuclear trafficking pathways.
5. Recognition of Donor and Target Compartments:
Recognition is crucial for appropriate assembly of transport complexes and cargo
release.
While β - karyopherins rely on the nucleotide state of the Ran GTPase for
recognition, the nature of this step is less clear for mRNA export, where extensive
mRNP remodeling is associated with both initiation of transport and cargo release.
Continued

6. Insights from Recent Work:
In budding yeast, the DEAD-box helicase Dbp5 facilitates the
removal of Mex67 after transport through NPCs.
The Dbp5 ATPase is activated by Gle1 and inositol hexaphosphate
(IP6), likely occurring when Dbp5 and Gle1 are bound to nucleoporins
at the NPC cytoplasmic face.

mRNA nuclear export involves the intricate remodeling of messenger
ribonucleoprotein complexes (mRNPs). As mRNA matures in the nucleus,
various proteins linked to gene-expression steps bind to it, with some
accompanying mature mRNPs to the cytoplasm.
Notably, the exon junction complex (EJC) plays a key role in nonsense-mediated
decay, and other proteins within mRNPs function in translation or mRNA
targeting.
While the precise recognition mechanism for mRNP maturation completion
remains unclear, Mex67:Mtr2 binding is crucial for export. Mex67, with an
mRNA-binding domain, relies on adaptor/accessory proteins such as
REF/Aly/Sub2, EJC components, or SR proteins for efficient binding to mRNPs.
This intricate collaboration highlights the complexity of mRNA export,
involving dynamic interactions during mRNP remodeling for the transition from
the nucleus to the cytoplasm.
Assembly of mRNP Export Complexes

mRNA Nuclear Export: A Complex Remodeling
Process
• mRNPs undergo intricate remodeling during nuclear export.
• Proteins associated with gene expression bind to maturing mRNA.
• Some proteins accompany mRNPs to the cytoplasm for various functions.
• EJC: Nonsense-mediated decay, translation, mRNA targeting.
• Mex67:Mtr2 binding is crucial for export, relies on adaptor proteins.

• mRNP Remodeling: As mRNA matures in the nucleus, various proteins associated with
gene expression become attached, forming a messenger ribonucleoprotein complex
(mRNP).
• Protein Functions: These proteins have diverse functions:
• EJC (Exon Junction Complex): Plays a role in nonsense-mediated decay,
translation, and mRNA targeting.
• Other Proteins: Function in translation or mRNA targeting within the cytoplasm.

• Mex67:Mtr2 Complex: Crucial for mRNA export, Mex67 relies on adaptor
proteins for efficient binding to mRNPs.Mex67: Has an mRNA-binding domain.
• Adaptor Proteins (e.g., REF/Aly/Sub2, EJC components, SR proteins): Facilitate
Mex67 binding to mRNPs

The movement of messenger ribonucleoprotein (mRNP) export complexes through nuclear pore
complexes (NPCs) relies on a Brownian ratchet mechanism, where the Mex67:Mtr2 heterodimer
interacts with FG-nucleoporins.
This mechanism aligns with the principles observed in nuclear trafficking pathways, emphasizing
passive diffusion and complex disassembly in the target compartment as key components.
The Brownian ratchet ensures the directionality of movement through the pores by preventing the
return of transport complexes to the donor compartment.
Despite the size disparity between mRNPs and cargoes transported by karyopherins, the Brownian
ratchet, lubricated by FG-nucleoporins, allows effective transport through NPCs. The removal of
molecules like Mex67:Mtr2 and other forms of mRNP remodeling can act as ratchet mechanisms,
preventing backward movement.
Translocation Through The Nuclear Pores

Brownian Ratchet Mechanism
• mRNP Export Complexes
• Movement through nuclear pore complexes (NPCs).
• Mex67:Mtr2 heterodimer interaction with FG-nucleoporins.
• Principles of Nuclear Trafficking
• Passive Diffusion: Observed in trafficking pathways.
• Complex Disassembly: Key components in the target compartment.

Ensuring Directionality
• Brownian Ratchet
• Directionality: Prevents return to donor compartment.
• Effective Transport: Despite size disparity with karyopherin cargoes.
• FG-Nucleoporins as Lubricants
• Facilitate transport through NPCs.
• Ratchet Mechanisms: Removal of Mex67:Mtr2 and mRNP remodeling.

For very large mRNPs, multiple ratchet steps may be necessary, each potentially employing
different mechanisms.
Moreover, the thermal ratchet mechanism could be complemented by pulling forces, possibly from
the translation machinery or cytoplasmic proteins.
In summary, the translocation of mRNP export complexes through NPCs is a sophisticated process
involving a blend of passive diffusion, Brownian ratchet mechanisms, and potential
complementary pulling forces, highlighting the intricate nature of cellular transport.
Continued

Figure 05 - Steps along the Gene-
Expression Pathway
The formation of a mature mRNP involves nuclear
processes and binding to the Mex67:Mtr2 complex.
This complex aids mRNP passage through nuclear
pores, interacting weakly with FG-nucleoporins.
Dbp5 at the NPC's cytoplasmic face may act as a
Brownian ratchet, removing Mex67:Mtr2 and
preventing mRNP return to the nucleus, ensuring
unidirectional transport.

Figure 06 - Schematic Illustration of How
a Brownian Ratchet Could Transport
Large mRNPs through NPCs
i) A mature mRNP likely contains multiple Mex67:Mtr2
complexes attached along its length. Interactions
between these complexes and FG-nucleoporins lining
the nuclear pore channel enable the mRNP to move
back and forth through thermal motion (Brownian
movement).
ii) A Mex67:Mtr2 complex reaching the NPC's cytoplasmic
face is removed by Dbp5, a DEAD-box helicase whose
ATPase activity is stimulated by Gle1 and IP6.
iii) The removal of Mex67:Mtr2 at the NPC's cytoplasmic
face acts as a molecular ratchet, preventing the
corresponding mRNP segment from moving back into
the transport channel.
iv) The removal of Mex67:Mtr2 prevents a longer mRNP
segment from returning, allowing iterative cycles. The
ATPase activity of Dbp5 rectifies thermal motion,
facilitating the large mRNP's movement into the
cytoplasm. Liberated Mex67:Mtr2 is recycled for
another export cycle.

mRNA export intricately involves the remodeling of mRNP complexes, with various proteins
associated with gene-expression steps accompanying mRNA.
Mex67:Mtr2 binding is crucial for export, though the exact mechanism recognizing completed
mRNP maturation remains unclear.
Translocating mRNP export complexes through NPCs relies on weak interactions with FG-
nucleoporins.
The transport occurs through simple diffusion, with Mex67:Mtr2 acting as a molecular Brownian
ratchet.
Despite mRNPs being larger, the smaller distances within NPCs, along with FG-nucleoporin
lubrication, enable effective transport.
Removal of Mex67:Mtr2

Removal of Mex67:Mtr2 from mRNPs, potentially acting as a molecular ratchet preventing nuclear
return, involves Dbp5, a DEAD-box helicase.
Dbp5 shows ATPase stimulation by Gle1 and IP6, with Gle1 serving as a spatial marker at the
cytoplasmic NPC face.
The Dbp5:Gle1:IP6 interaction is crucial for mRNA export, likely involving conformational
changes and providing directionality through the NPC.
The roles of Gle1, IP6, and Dbp5 in budding yeast highlight their importance in mRNA export.
The conservation of these factors in metazoans suggests a strongly preserved mRNA export
mechanism across different organisms.
Continued

While this model aligns with recent observations, the evidence for certain steps
in this process is either lacking or indirect.
The significance of Dbp5-mediated Mex67 release and Gle1:IP6 stimulation of
Dbp5 ATPase for mRNP export is emphasized.
However, it is noted that direct demonstration of these events at the cytoplasmic
face of the nuclear pore complex (NPC) is yet to be established.
The binding of Gle1 and Dbp5 to specific nucleoporins (Nup42 and Nup159,
respectively) strongly suggests that the stimulation occurs at the NPC
cytoplasmic face, although direct confirmation is still pending.
Future Directions

Several crucial aspects remain unclear, such as the molecular basis for Mex67 attachment and
removal, the involvement of other mRNP-bound proteins (e.g., EJC and SR proteins), and the
quantities of these proteins on export-competent mRNPs.
Additionally, it is uncertain whether the Gle1:IP6-stimulated release of Mex67 by Dbp5 is the sole
step required for mRNA export or if it is just one of several mRNP-remodeling processes involved.
The passage raises questions about how Dbp5 locates Mex67 on the mRNP, the potential role of
other proteins like Nab2 and Gfd1, and whether the activity of Dbp5 can modify mRNP structure
over a considerable distance.
The complexity of the mRNA export process is acknowledged, and the need to decipher precisely
how Gle1, IP6, and Dbp5 contribute to mRNP export, alongside other potential remodeling
processes, is recognized as a challenging yet promising endeavor.
The ultimate goal is to gain profound insights into the intricate mechanisms governing mRNA
export.
Continued

Glucose Transport Across Plasma Membranes: Facilitated
Diffusion Systems
By Stephen A. Baldwin and Gustav E. Lienhard.

Glucose transport across plasma membranes involves facilitated diffusion systems.
Here's a simplified explanation of the process:
1. **Transport Proteins**: - Glucose transporters, such as GLUT proteins, are integral membrane
proteins responsible for facilitating the movement of glucose across the plasma membrane.
2. **Facilitated Diffusion**: - Facilitated diffusion is a passive transport mechanism where glucose is
moved from an area of high concentration to an area of low concentration without requiring
energy input.
3. **GLUT Proteins**: - GLUT proteins undergo conformational changes to transport glucose. When
glucose binds to the transporter on one side of the membrane, the protein changes shape, allowing
glucose to be released on the other side of the membrane.
Introduction

4. **Specificity**: - GLUT proteins exhibit specificity for glucose and related
sugars, ensuring that only glucose molecules are transported across the
membrane.
5. **Regulation**: - The activity of glucose transporters can be regulated to control
the rate of glucose uptake based on the cell's metabolic needs or external glucose
levels.
6. **Role in Metabolism**: - Glucose transport across membranes is essential for
providing cells with a constant supply of glucose, which serves as a primary
energy source for cellular processes like glycol sis and ATP production.

In the context of Glucose Transport Across Plasma Membranes through Facilitated
Diffusion Systems, the following events occur:
 **Glucose Binding**: - Glucose molecules bind to specific glucose transporter
proteins embedded in the plasma membrane.
 **Conformational Changes**: - Upon glucose binding, the transporter protein
undergoes conformational changes, allowing the glucose molecule to be transported
across the membrane.
 **Cellular Utilization**: - Once inside the cell, glucose can be utilized in various
metabolic pathways, such as glycolysis, to generate energy (ATP) for cellular
processes.

Identification and Isolation of Glucose Transporter
Identification and isolation of glucose transporter proteins involve several steps:
1. **Cell Extraction**: - Cells containing the target glucose transporter protein are
collected and lysed to release cellular contents.
2. **Membrane Fractionation**: - The cell lysate is subjected to membrane
fractionation techniques to isolate the plasma membrane components from the
rest of the cell contents.
3. **Protein Purification**: - Various purification methods, such as chromatography,
are employed to isolate the specific glucose transporter protein from other
membrane proteins.
4. **Protein Identification**: - Techniques like mass spectrometry are used to
identify the purified protein and confirm its identity as the glucose transporter.
5. **Characterization**: - The isolated glucose transporter protein is characterized
for its structure, function, and regulatory mechanisms to understand its role in
glucosetransport.

Structure of The Isolated Transporter
When examining the structure of an isolated transporter protein, especially in the context of
glucose transporters, several aspects are typically considered:
**Membrane Spanning Regions**: - Transporter proteins usually have multiple transmembrane
domains that span the lipid bilayer of the cell membrane. These regions create a channel for the
passage of molecules like glucose.
**Binding Sites**: - Within the protein structure, specific binding sites interact with glucose
molecules to facilitate their transport across the membrane.
**Functional Domains**: - Different domains within the transporter protein contribute to its
overall function in transporting glucose or other substrates.

When considering the functional properties of an isolated transporter protein, such as a glucose
transporter, several key aspects are typically examined:
The transporter's specificity for glucose as the substrate it transports across the membrane.
Understanding how the transporter facilitates the movement of glucose molecules across the
membrane, often through facilitated diffusion. Investigating how the activity of the transporter is
regulated in response to cellular signals or changes in glucose concentration.
Exploring potential inhibitors that can block the function of the transporter, providing insights into
its mechanism of action. Determining if the transporter operates alone or in conjunction with other
molecules to transport glucose into or out of the cell.
Examining where the transporter is localized within the cell and how this impacts its function in
glucose transport.
Functional Properties of Isolated Transporter

The mechanism of glucose transport through the membrane involves a process where glucose
molecules are moved across the cell membrane. Here's a simplified explanation:
 Glucose transporters are proteins embedded in the cell membrane that facilitate the movement of
glucose molecules into or out of the cell.
 These transporters undergo conformational changes, where they switch between different shapes
to transport glucose across the membrane.
 The transporters have specific binding sites for glucose molecules. When a glucose molecule
binds to the transporter, it triggers a change in shape that allows the molecule to be transported
across the membrane.
 Competitive inhibition studies, where other molecules compete with glucose for binding to the
transporter, provide evidence for how the process works.
The Mechanism of Transport

Insulin stimulation of glucose transport in adipocytes and muscle cells involves specific mechanisms
that enhance the movement of glucose into these cells.
 **Similar Transport Systems**: Adipocytes and muscle cells have glucose transport systems
similar to the transporter found in human erythrocytes.
 **Effect of Insulin**: Studies in rat adipocytes indicate that insulin stimulation increases the
maximum velocity (Vmax) for glucose transport without affecting the Michaelis constant (Km).
This means that insulin enhances the rate at which glucose is transported into the cells without
changing the affinity of the transporter for glucose.
 **Increased Transporter Quantity**: Using cytochalasin B binding assays, researchers have
shown that insulin increases the number of transporters in the plasma membrane. This increase in
transporter quantity contributes to the enhanced glucose transport facilitated by insulin.
Insulin Stimulation of Transport

Similar to the impact of insulin, the transformation of chick embryo fibroblasts by Rous sarcoma
virus results in a significant increase in the maximum velocity (Vmax) for hexose transport,
without altering the Michaelis constant (Km).
Utilizing cytochalasin B binding for quantification, Salter and Weber establish a direct
proportionality between the rise in transport rate and the increase in the number of transporters in
the plasma membrane.
However, in this case, the heightened transport rate is largely contingent on protein synthesis,
indicating a different mode of stimulation compared to insulin in adipocytes.
Transformation and Transport

Further advancements in the understanding of the human erythrocyte glucose transporter are anticipated
with its availability in a pure form and substantial quantities.
 The focus will extend to the structural aspects of the polypeptide chain, particularly its folding within the
lipid bilayer, and the specific regions implicated in the conformational changes associated with the
translocation step.
 The methodologies developed for the purification of the erythrocyte transporter are poised to be valuable
tools in isolating similar transporters from various cell types, including adipocytes, fibroblasts, brain
cells, liver cells, and muscle cells, all of which exhibit facilitated diffusion systems for D-glucose.
 This opens up possibilities for in-depth investigations into the mechanisms of control, especially in cell
types where glucose transport is subject to regulation.
 As purified transporters become available from different cell types with regulated transport, researchers
can employ a variety of approaches to delve into the intricate mechanisms governing transporter function
and regulation.
Prospects For The Future

Thanks

Advanced Cell Biology good and best ppt on the mrna transport (1).pptx

Recommandé

Recommandé

Contenu connexe

Similaire à Advanced Cell Biology good and best ppt on the mrna transport (1).pptx

Similaire à Advanced Cell Biology good and best ppt on the mrna transport (1).pptx (20)

Plus de GashawDesta2

Plus de GashawDesta2 (8)

Dernier

Dernier (20)

Advanced Cell Biology good and best ppt on the mrna transport (1).pptx

Notes de l'éditeur