Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps

Haiyan Chu, Estela Puchulu-Campanella, Jacob A. Galan, W. Andy Tao, Philip S. Low, Joseph F. Hoffman

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

The type of metabolic compartmentalization that occurs in red blood cells differs from the types that exist in most eukaryotic cells, such as intracellular organelles. In red blood cells (ghosts), ATP is sequestered within the cytoskeletal-membrane complex. These pools of ATP are known to directly fuel both the Na+/K+and Ca2+ pumps. ATP can be entrapped within these pools either by incubation with bulk ATP or by operation of the phosphoglycerate kinase and pyruvate kinase reactions to enzymatically generate ATP. When the pool is filled with nascent ATP, metabolic labeling of the Na+/K+ or Ca2+ pump phosphoproteins (E Na-P and ECa-P, respectively) from bulk [γ- 32P]-ATP is prevented until the pool is emptied by various means. Importantly, the pool also can be filled with the fluorescent ATP analog trinitrophenol ATP, as well as with a photoactivatable ATP analog, 8-azido-ATP (N3-ATP). Using the fluorescent ATP, we show that ATP accumulates and then disappears from the membrane as the ATP pools are filled and subsequently emptied, respectively. By loading N3-ATP into the membrane pool, we demonstrate that membrane proteins that contribute to the pool's architecture can be photolabeled. With the aid of an antibody to N3-ATP, we identify these labeled proteins by immunoblotting and characterize their derived peptides by mass spectrometry. These analyses show that the specific peptides that corral the entrapped ATP derive from sequences within β-spectrin, ankyrin, band 3, and GAPDH.

Original languageEnglish (US)
Pages (from-to)12794-12799
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number31
DOIs
StatePublished - Jul 31 2012

Keywords

  • Confocal microscopy
  • Membrane peptides
  • Western blots

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps'. Together they form a unique fingerprint.

Cite this