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Identification of protein complexes with quantitative proteomics in S. cerevisiae
Published on: March 4, 2009
Immunopurification of a sarcomeric junctional protein complex containing GAPDH
K A McDonald1, J Muschler, A F Horwitz
1Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA. kmcdonal@uiuc.edu
Insights
Researchers identified a novel protein complex containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at muscle and nerve anchorage junctions. This complex may reinforce cytoskeletal structures.
Area of Science:
- Cell Biology
- Neuroscience
- Muscle Physiology
Background:
- Anchorage junctions are critical for cell structure and function.
- The molecular composition of these junctions, particularly at neuromuscular junctions and muscle Z-disks, is incompletely understood.
- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is primarily known as a glycolytic enzyme but has emerging roles in non-metabolic cellular processes.
Purpose of the Study:
- To characterize a novel protein complex localized to anchorage junctions.
- To identify the molecular components of this complex, particularly those associated with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
- To investigate the role of this complex in muscle development and cytoskeletal organization.
Main Methods:
- Isolation and characterization of a monoclonal antibody (P4B2) targeting anchorage junctions.
- Immunopurification of the P4B2 antigen from chicken brain tissue.
- Protein identification using amino acid sequencing and SDS-PAGE.
- Immunolocalization studies in developing cultured muscle cells.
Main Results:
- The monoclonal antibody P4B2 localized to focal adhesions, muscle Z-disks, and neuromuscular junctions.
- Immunopurification revealed a protein complex consisting of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 36 kDa) and proteins associated with the synaptic vesicle fusion core complex (30 and 18 kDa).
- In developing muscle cells, the P4B2 antigen was incorporated into Z-lines concurrently with alpha-actinin, indicating its role in sarcomeric organization.
Conclusions:
- A novel protein complex containing GAPDH and synaptic vesicle proteins exists at multiple anchorage junctions.
- This GAPDH-containing complex is associated with the Z-disk in muscle cells, suggesting a role in reinforcing cytoskeletal structures.
- The findings suggest a non-glycolytic function for GAPDH in cellular architecture and signaling at specialized cell junctions.
Abstract:
We have isolated a monoclonal antibody, P4B2, which localizes to multiple anchorage junctions, namely, a subset of focal adhesions, the Z-disk of muscle, and neuromuscular junctions. Immunopurification of the antigen to this antibody from chicken brain tissue yielded a complex of three prominent proteins with mobilities of 36, 30, and 18 kDa. Amino acid sequencing of the purified proteins identified the 36-kDa protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other two protein bands were heterogeneous, containing proteins found in the synaptic vesicle fusion core complex. Immunolocalization of P4B2 antigen in developing cultured muscle cells showed that the antigen is incorporated into Z-lines soon after the sarcomeric architecture was positive for alpha-actinin. Together, the data indicate the P4B2 antigen is part of a unique GAPDH-containing protein complex that may be involved in reinforcement of established cytoskeletal structures.
