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However, lipid metabolism in spermatozoa is not clearly understood for two reasons: i) the mature spermatozoa are devoid of endoplasmic reticulum, which is the major site of phospholipid (PL) synthesis in somatic cells, and ii) studies have been superficial due to the difficulty in culturing spermatozoa. We hypothesize that spermatozoa contain biogenic membrane flippases since immense changes in lipids occur during spermatogenic differentiation. To test this, we isolated spermatozoa from bovine epididymides and reconstituted the detergent extract of sperm membranes into proteoliposomes. In vitro assays showed that proteoliposomes reconstituted with sperm membrane proteins exhibit ATP-independent flip\u2013flop movement of phosphatidylcholine (PC), phosphatidylserine, and phosphatidylglycerol. Half-life time of PC flipping was found to be \u223c3.2\u00b11\u200amin for whole sperm membrane, which otherwise would have taken \u223c11\u201312\u200ah in the absence of protein. Further biochemical studies confirm the flip\u2013flop movement to be protein-mediated, based on its sensitivity to protease and protein-modifying reagents. To further determine the cellular localization of flippases, we isolated mitochondria of spermatozoa and checked for ATP-independent flippase activity. Interestingly, mitochondrial membranes showed flip\u2013flop movement but were specific for PC with half-life time of \u223c5\u00b12\u200amin. Our results also suggest that spermatozoa have different populations of flippases and that their localization within the cellular compartments depends on the type of PL synthesis.<\/jats:p>","DOI":"10.1530\/rep-13-0121","type":"journal-article","created":{"date-parts":[[2013,6,25]],"date-time":"2013-06-25T23:13:39Z","timestamp":1372202019000},"page":"209-220","update-policy":"https:\/\/doi.org\/10.1530\/crossmarkpolicy-1","source":"Crossref","is-referenced-by-count":1,"title":["Biochemical evidence for energy-independent flippase activity in bovine epididymal sperm membranes: an insight into membrane biogenesis"],"prefix":"10.1093","volume":"146","author":[{"given":"Archita","family":"Rajasekharan","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vincent Gerard","family":"Francis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sathyanarayana N","family":"Gummadi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2013,9,1]]},"reference":[{"key":"2026010712260584400_bib1","doi-asserted-by":"crossref","first-page":"724","DOI":"10.1016\/j.bbrc.2003.08.065","article-title":"A hierarchy of lipid constructs for the sperm plasma membrane","volume":"309","author":"Apel-Paz","year":"2003","journal-title":"Biochemical and Biophysical Research Communications"},{"key":"2026010712260584400_bib2","doi-asserted-by":"crossref","first-page":"9843","DOI":"10.1021\/la050568r","article-title":"Impact of membrane cholesterol content on the resistance of vesicles to surfactant attack","volume":"21","author":"Apel-Paz","year":"2005","journal-title":"Langmuir: The ACS Journal of Surfaces and Colloids"},{"key":"2026010712260584400_bib3","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1038\/327341a0","article-title":"Reconstitution of a phospholipid flippase from rat liver microsomes","volume":"327","author":"Backer","year":"1987","journal-title":"Nature"},{"key":"2026010712260584400_bib4","doi-asserted-by":"crossref","first-page":"524","DOI":"10.1002\/prca.200601013","article-title":"Identification of gene products present in Triton X-100 soluble and insoluble fractions of human spermatozoa lysates using LC\u2013MS\/MS analysis","volume":"1","author":"Baker","year":"2007","journal-title":"Proteomics. 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