Unit 03 pt1

Unit 3 Part 1 Overview

• Introduction to cell compartments

• Review of topics covered in previous units

• Emphasis on microscopy techniques, including transmission electron micrographs

Organelles and Cell Compartments

Visualization of Compartment Volumes

• Table 1: Volumes and ratios of organelles in a typical eukaryotic cell (e.g., hepatocyte)

  • Cytoplasm > 50% of cell volume

  • Mitochondria significant, smaller organelles occupy less volume

• Table 2: Membrane ratios in different cell types (e.g., hepatocyte vs. pancreatic exocrine cell)

  • Hepatocyte: High rough endoplasmic reticulum (ER) and mitochondria for metabolism

  • Pancreatic beta cell: Rough ER represents 60% of total cell membrane for insulin production

Evolutionary Origins of Organelles

Classification of Organelles

• Organelles can be categorized based on evolutionary origins

• Category 1: Nucleus and cytoplasm evolved together

• Category 2: Endomembrane system organelles (e.g., ER, Golgi apparatus) from a common event

• Category 3: Mitochondria and chloroplasts emerged from different endosymbiotic events

• Key takeaway: Organelles categorized into four groups based on evolutionary history

Protein Synthesis and Sorting

Protein Synthesis Overview

• Initial synthesis occurs in the cytosol for most proteins

• Exception cases exist where proteins are synthesized directly in organelles

Signal Sequences

• Amino acid sequences direct proteins to their destinations, known as sorting signals

• Proteins lacking sorting signals remain in the cytoplasm

• Organelles cannot form de novo; they rely on information from existing organelles

Modes of Protein Sorting

Categories of Protein Sorting

1. Gated transport: Nuclear pore complexes transport macromolecules; small molecules diffuse freely

2. Transmembrane transport: Proteins are directly transported across membranes, typically in an unfolded state

3. Vesicular transport: Transport vesicles carry soluble and membrane proteins to new compartments

4. Engulfment: Membrane encloses materials (e.g., phagocytosis)

• Figures illustrate vesicle transport and membrane protein orientation

Signal Sequences Details

Structure and Function

• Signal sequences: 15-60 amino acids, often at N-terminus, cleaved upon delivery

• Exception cases: Internal sequences or carboxyl-terminal sequences can also act as signals

• Signal patch: 3D configuration aiding in directing proteins; may not be part of primary sequence

• Example: Mannose-6-phosphate directs proteins to lysosomes

Examples of Signal Sequences

• Expected to recognize:

  • Basic amino acid clusters for nuclear import

  • KDEL sequence for ER retention

Receptors in Protein Sorting

• Receptors recognize sorting sequences, guide proteins to destinations, and can be recycled

• Common designations of amino acids and their 3-letter and single-letter codes are provided for reference

Experimental Validation of Signal Sequences

Techniques

• Use of recombinant DNA to attach signal sequences to proteins

• Transfect cells to express fusion proteins, utilizing techniques like immunofluorescence and cell fractionation to verify localization

• Site-directed mutagenesis can reveal the importance of specific amino acid sequences

Subcellular Fractionation Techniques

Cell Disruption Methods

• Techniques include osmotic pressure, sonication, or mechanical disruption (e.g., French press, grinding)

• Resulting cell extract (homogenate) undergoes centrifugation to separate components

Centrifugation Process

• Series of centrifugation steps to isolate specific cellular components

• Pellets formed at each stage represent different organelles (e.g., mitochondria, lysosomes)

Analytical Techniques

Methods of Characterization

• Use of SDS PAGE and western blotting for identification of cell components

• Assays for enzymatic activity, focusing on cytochrome oxidase (COX) in isolated fractions

• Hands-on experience isolating subcellular fractions from yeast cells

Final Notes

• This concludes the overview for Unit 3 Part 1, further details and implications will be explored in subsequent videos.