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

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

2. 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

3. Protein Transport Across the Endoplasmic Reticulum By Tom A. Rapoport B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 3

4. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 4

5. 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 B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 5

6. 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 B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 6

7. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 7

8. 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 B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 8

9. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 9 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.

10. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 10 Structure and Function of Translocation Channel

11. ** 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 11 Key Features of the Archeal SecY Complex Structure

12. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 12

13. In the translocation process, several key points occur to facilitate the movement of proteins across cellular membranes. Here are some important steps: **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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 13 Key points in the translocation process

14. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 14 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.

15. 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: **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 translocation process. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 15 Oligomeric Translocation Channels

16. The Sec61/ SecY complex likely forms oligomers during co-translational translocation. Here's a breakdown of the process: **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 . B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 16 Key Points Regarding The Involvement of Oligomers in Translocation

17. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 17 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 translocation process. 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.

18. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 18 Membrane Protein Integration

19. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 19 Continued

20. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 20 Maintaining The Permeability Barrier

21. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 21 Continued

22. Ratcheting mRNA Out of The Nucleus By Stewart M. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 22

23. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 23 Introduction

24. Con.t These pathways involve three key steps: T he generation of a cargo:carrier complex in the donor compartment , T ranslocation of this complex through nuclear pore complexes ( npcs ) , and The release of cargo in the target compartment , F ollowed by the recycling of the carrier. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 24

25. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 25 Key Points About nuclear trafficking pathways, Including mRNA Export

26. 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 . B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 26 Continued

27. 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. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 27

28. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 28 Assembly of mRNP Export Complexes

29. 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. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 29

30. 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 . B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 30

31. 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 B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 31

32. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 32 Translocation Through The Nuclear Pores

33. 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. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 33

34. 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. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 34

35. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 35 Continued

36. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 36 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.

37. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 37 Figure 06 - Schematic Illustration of How a Brownian Ratchet Could Transport Large mRNPs through NPCs 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). 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. 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. 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.

38. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 38 Removal of Mex67:Mtr2

39. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 39 Continued

40. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 40 Future Directions

41. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 41 Continued

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43. Glucose Transport Across Plasma Membranes: Facilitated Diffusion Systems By Stephen A. Baldwin and Gustav E. Lienhard . B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 43

44. Glucose transport across plasma membranes involves facilitated diffusion systems. Here's a simplified explanation of the process: **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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 44 Introduction

45. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 45 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.

46. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 46 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.

47. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 47 Identification and Isolation of Glucose Transporter I dentification and isolation of glucose transporter proteins involve several steps: **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**: - V arious 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 .

48. 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. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 48

49. 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. B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 49 Functional Properties of Isolated Transporter

50. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 50 The Mechanism of Transport

51. 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**: S tudies 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 51 Insulin Stimulation of Transport

52. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 52 Transformation and Transport

53. 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. B.B.Tefera , G.D.Yimer & R.G.Sija , 2024 © 53 Prospects For The Future

54. Thanks B.B.Tefera, G.D.Yimer & R.G.Sija, 2024 © 54