Analysis of transmembrane proteins from eukaryotic cells

Area of Science:

• Cell Biology
• Biochemistry
• Membrane Protein Topography

Background:

• Understanding plasma membrane protein organization is crucial for cell function.
• Enzymatic labeling offers a method to probe protein accessibility on membrane surfaces.

Purpose of the Study:

• To determine the topographic organization of plasma membrane proteins in mouse L-929 cells.
• To utilize phagolysosomes as an 'inside-out' membrane preparation for studying protein accessibility.

Main Methods:

• Enzymatic labeling (lactoperoxidase-catalyzed iodination) of external and internal membrane surfaces.
• Preparation of phagolysosomes from latex-particle-ingesting cells.
• Two-dimensional gel electrophoresis to analyze labeled proteins.
• Partial hydrolysis and phosphorylation studies (using 32P).

Main Results:

• Phagolysosomes exhibit an 'inside-out' orientation of the plasma membrane.
• Specific membrane glycoproteins (60,000 and 100,000 daltons) are accessible for labeling on both membrane faces.
• Different peptides of the 100,000 dalton protein are iodinated from external versus internal surfaces.
• Identified proteins are extensively phosphorylated.

Conclusions:

• Phagolysosomes serve as a valuable model for studying membrane protein topography.
• Evidence suggests specific plasma membrane proteins span the lipid bilayer.
• The study defines the accessibility and potential transmembrane nature of key membrane proteins.