Secretory Pathway 1 & 2

Transport in the Secretory Pathway

Key Locations:

  • Endoplasmic Reticulum (ER): The site of protein synthesis and initial folding.

  • Golgi Apparatus (GA): Responsible for post-translational modifications, sorting, and distribution of proteins.

  • Pre-Golgi: Comprises structures such as vesicular-tubular clusters (VTCs) which connect the ER and Golgi.

  • Cell Surface/Secretory Granules/Endosomes: Final destinations for secreted proteins or recycling hubs.

Organelles Involved

  • Microtubule Organizing Center (MTOC): Facilitates the polymerization of microtubules, providing tracks for vesicular transport.

  • Centrioles: Assist in microtubule organization and are crucial for proper cell division.

Vesicular Trafficking Steps

  1. Sorting of Cargo: Determining the specific proteins that require transport based on signals within their sequences.

  2. Budding: Vesicles form from donor membranes via a process involving coat proteins that help in shaping the vesicles (e.g., COPII at the ER).

  3. Separation from Source Membrane: The vesicle detaches from the ER or Golgi, involving dynamin, a GTPase that constricts the neck of the budding vesicle.

  4. Transfer: The vesicle is transported to its destination, often aided by motor proteins along microtubules.

  5. Storage: Some proteins can be stored in granules, especially in neurons where synaptic vesicles hold neurotransmitters.

  6. Recognition and Fusion: Proteins on the vesicle (v-SNAREs) interact with target membrane proteins (t-SNAREs) to dock and fuse, releasing the cargo.

Formation and Structure of COPII Vesicles

  • At a high enough concentration, COPII forms cage structures with itself

    • COPII naturally wants to be curved

  • COPII subunits are found in the cytoplasm

  • They must be brought to ER membranes to assemble and form a coat

  • They must then be released after vesicle budding is completed

  • Structural components:

    • Sar1: A small GTPase essential for COPII coat assembly and disassembly, acting as a switch for vesicle formation.

      • In the cytoplasm bound to GDP

      • Attached to the membrane bound to GTP

        • Recruits Sec23/24 and binds

        • Contains a hydrophobic sequence that interacts with the hydrophobic sequence of the membrane

      • GEF is Sec12, brings Sar1 to the membrane and acts as a GEF, exchanging GDP for GTP

        • Found in the ER membrane at ER exit sites (where vesicle formation is happening)

    • Inner Subunits (Sec23/24): Recognize signals on membrane proteins and bind cargo proteins during vesicle budding.

      • Two phenylalanines (F) at the C-terminus of protein substrates.

      • A cluster of acidic amino acids in the cytoplasmic domain of transmembrane proteins ensures specificity in COPII binding.

      • Once bound, recruit Sec 13/31

    • Outer Subunits (Sec13/31): Generate membrane curvature, crucial for facilitating vesicle budding.

      • Once bound, enforce a conformational change in Sec23/24, which causes Sar1 to hydrolyze its ATP, destabilizing the complex, causing it to leave the membrane

        • The coat subsequently disassembles and the uncoated vesicle fuses with VTC membranes

    • Pre-Golgi Intermediate: Complex of tubules, often tipped with COPI coats

      • Formation:

        • The golgin tethering factor p115, brought in by Rab1 (bounded to GTP), tether to COPII vesicles

        • These tethered vesicles then fuse together in an NSF and SNARE-dependent mechanism to form VTCs

      • These vesicles utilize the cytoskeleton, moving along microtubules with the assistance of dynein (retrograde transport) and kinesin (anterograde transport) motor proteins.

      • COPI vesicles containing cargo receptors bud off of VTC and fuse with nearby ER returning empty cargo receptors and some other retrograde cargo back to ER

Discovery of COPII Vesicles

  • Centrifugation:

    • Differential centrifugation is achieved by subjecting a suspension of components (disrupted cells) to centrifugal forces, and the heavier components will reach the bottom first

    • Density gradient centrifugation is an improvement on differential centrifugation in which a sucrose or metrizamide gradient is established. The gradient involved greater density at the bottom and lower density at the top. The components added will rest at the level of equal density.

      • Smooth gradients made by machine

      • Stub gradient made by layering solutions with different concentrations (densities)

    • Differential centrifugation is used to pull out the nuclei and another step to get rid of mitochondria and lysosomes. Then what is left, is placed on the sucrose gradient and allowed to equilibrate

  • Schekaman’s isolation of temperature-sensitive Sec:

    • Yeast cells were exposed to a mutagen to induce genetic mutations.

    • They were then grown at 24°C.

    • The yeast cells were then shifted to a higher temperature (37°C) for three hours.

    • This allowed identification of temperature-sensitive mutants—cells that failed to secrete proteins at 37°C.

    • These "extra-heavy" yeast cells (due to accumulated proteins) were separated using density gradient centrifugation.

    • The yeast was moved back to 24°C so as not to block secretion for too long and kill the cells.

    • Colonies were screened to identify secretion-defective mutants.

    • Mutations in 23 different genes were initially identified.

  • Complementation tests:

    • Mutant haploid yeast were mated to from diploid progeny in a complementation test to determine which mutations ware on the same gene

    • Mating mutations on the same gene → no complementation; mutated phenotype

    • Mating mutations on different genes → complementation; wild-type phenotype

    • The 23 complementation groups were sorted depending on whether ER accumulates at non-permissive temperature (suggesting block in exit from ER), golgi membrane accumulated (enlarged yeast golgis were referred to as “Berkeley bodies”), or whether vesicles accumulated

    • In accumulates vesicles, it was assumed that the proteins coded by these genes were required to fuse with plasma membrane after leaving the golgi

  • Supressor mutations:

    • If protein has reduced function because of mutation, mutations in interacting proteins will sometimes restore function

    • In other cases, overespression of a protein operating in the same of parallel pathway can sometimes rescue function

    • Sar1 was identified because it rescued the sec12 mutation when overexpressed

  • Synthetic lethality:

    • When yeasts carrying two different mutations are mated, the progeny have double mutations. Synthetic lethality is when the presence of both of these proteins causes lethality

    • Through this, Schekaman determined Sec13/31 and 23/24 interact

  • Discovery of COPII:

    • Vesicles could be made from purified ER by incubating the ER with cytosol, ATP, GTP, and some other factors

    • Coated vesicles could be accumulated when a GTP analog was introduced which could not be hydrolyzed, i.e. Sar1 is stuck in the membrane

Cargo Receptors

  • Function: Cargo proteins lacking transmembrane domains must associate with cargo receptors, which remove them from the ER and mediate their incorporation into COPII vesicles.

  • Recycling: After delivering cargo, these receptors are recycled back to the ER via retrograde pathways, essential for maintaining cellular homeostasis.

  • ERGIC53 Example: ERGIC53 is a cargo receptor that binds to N-linked oligosaccharides

  • Many cargo receptors have the ability to interact with another coat (COPI)

    • The most common interaction motif is lysine pairs followed by other amino acids (e.g., ERGIC53 ending in -KKFF). This is recognized by COPI and COPII, facilitating the transport of soluble proteins.

Mechanisms of Vesicle Movement and Fusion

  • Microtubule Dynamics: Dynein transport pre-golgi intermediates to the golgi on microtubules

  • Fusion with cis-golgi:

    • Step 1 - tethering

      • p115 is one tethering protein that is involved, it binds to the small GTPase Rab1

    • Step 2 - SNARE proteins interact

      • t-SNAREs work on the target membrane

      • v-SNAREs on the vesicle membrane

      • The right t and v SNAREs have to interact for membrane fusing

    • Step 3 - membranes fuse

Golgi to ER (Retrograde) Trafficking

  • Some proteins sent from the ER to the Golgi (cargo receptors, v-SNAREs, etc.) must be recycled back to the ER for reuse

  • There is therefore a reverse pathway from the golgi to ER, it is much more poorly understood than ER to golgi trafficking'

  • Pathway 1: COPI dependent retrograde trafficking employs COPI-coated vesicles

  • Pathway 2: COPI independent pathway involves the formation of tubular transport intermediates and does not require COPI

Super-Resolution Microscopy

Near-Field Microscopy

  • Move optical fiber with 30 nm tip slowly over the sample

  • Evaded diffraction limit and enabled super-resolution fluorescence imaging

  • Image acquisition was slow (30 min/image)

  • Impractical for biological use, okar for materials science

STORM

  • Stochastic Optical Reconstruction Microscopy

  • The sample is placed in a special buffer that drives most of the fluorescent dye molecules into a dark state

  • The few remaining molecules are distant from each other and can be individually visualized (with intense illumination and a very good camera)

  • Molecules randomly flicker into and out of dark state. Images are taken (about 10/second) for approximately 15 minutes. This allows eventual visualization of most of the molecules, localizing them to ~30 nm

  • An image can be assembled showing all the molecule locations

PALM

  • Photoactivation Localization Microscopy

  • Similar to STORM but works with special photoactivaatable GFPs

  • A small proportion of paGFP can be turned on by near UV light, imaged, and bleached. Others can be turned on after, with the process repeated ~!000 times

  • First point-localization technique. Principle and result very similar to STORM

PALM/STORM limitations

  • Currently, not compatible with confocal microscopy

  • Best with very thin samples so single fluroescent molecules arent mistaken for autofluorescence. Use of TIRF can help, but limits observation to the bottom of samples

  • Often used on sections prepared as for EM. Can substititute for immunogold if you are willing to image the same section on two microscopes

  • Very difficult with living cells

Other Super-Resolution Techniques

  • Structured illumination

    • Doubles illumination

    • Not as difficult

  • STED

    • Works with confocal

  • AiryDisk

    • Works with confocal

    • Easier than STED