Membrane‐bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins

Abstract

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference lo the fractions of these proteins which are membrane.bound. In addition to EGTA.extmetable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilizcd whh detergents (Triton X-100 or Triton X-114). A strong base like Na:CO~, which is usually effective in extracting peripheral membrane proteins, only partially solubilizes the membranebound. EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble ann¢xins partition in the aqueous phase, the remaining fraetion~ being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as ),et unknown mechanism, following Ca:'-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of tbe~e proteins remain bound to membranes in a Ca:*-indepcndent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.Annexin V (CaBP33 and CaBP37); Annexin Vl; Membrane; Binding; Calcium; Lun~g; Heart; Brain I, INTRODUCTIONProteins of the annexin family share the ability to bind to acidic phospholipids and to natural membranes in the presence of Ca:* (for reviews see [1][2][3]). Each annexin is made of an N-terminal tail of variable length and unique to individual annexins, which is supposed to play a role in the diversification of the biological functions of single species, and of a core. This latter is made of four, in the ease of the 32-37 kDa annexins, or eight, in the case of the 67-73 kDa annexins, internal repeats 70 residues in length, each of which contains a highly conserved consensus sequence, the endonexin fold, which is suggested to take part in the coordination of binding of both Ca 2+ and phospholipids [3][4][5]. Unlike the Ca-'+-binding pro~:eins of the EF-hand type, Ca-'*-binding to annexins does not induce the exposure of hydrophobic domains; rather~ Ca 2. would cross-bridge any annexin to the negatively charged headgroups of acidic phospholipids and/or certain annexins to target proteins [1][2][3]. Given the above model, it is expected that chelation of Ca 2÷ will result in the complete reversal of annexin binding. While this has been proven true whenever annexin binding to liposomes or proteins had been investigated in reconstitution experiments in vitro [6][7][8][9][10][11][12][13][14][15], different results were obtained with certain annexins whenever tissue or cell subfractionation was used to investigate the binding of endogenous annexins to natural membranes. So, fractions ofannexins I, 1I, IV-VII were reported to exist in several cell types in a membrane-bound form, to resist extraction with EGTA, and to require detergents for their solubilization [10,[16][17][18][19][2...

Relocation of annexin V to platelet membranes is a phosphorylation-dependent process

“…In addition, subcellular fractionation studies have shown that in heart, lung, liver, platelets and brain a proportion of the total annexin V remains associated with membranes even after extraction with EGTA. It is therefore likely that annexin V plays a role in the regulation of a membranelocalized process [7,8,[25][26][27][28]. We have demonstrated that, following physiological platelet activation, annexin V relocates from the cytosol and binds to membranes, which implicates annexin V in coupling increases in cytosolic [Ca# + ] to membranespecific processes [7,8].…”

Involvement of annexin V in the entry of influenza viruses and role of phospholipids in infection

“…The cruvirus-binding protein we found probably corresponds to ancial question is whether binding of influenza virus to annexin nexin V. Further experiments showed that infection of influ-V (or annexin 33 kDa) is an essential step for infection. Anenza viruses could be inhibited by externally added phosphonexins are thought to be both intracellular and plasma memlipids as well as by antiserum against annexin V. These results brane components [10][11][12]. If the binding of viral phospholisubstantiated our view that annexins may serve as a second pids to the annexins of the plasma membrane is an essential receptor for these viruses, step for infection, externally added phospholipids should compete in this reaction, resulting in the inhibition of virus infec-2.…”

“…brane components [10][11][12]. To test the effect of anti-annexin V antiserum on virus multiplication, it was added to cells before viruses were given and the cells were maintained in the constant presence of this antiserum.…”

DT40 cells lacking the Ca 2+ -binding protein annexin 5 are resistant to Ca 2+ -dependent apoptosis

“…Both Anx2 and Anx5 are widely expressed, although Anx5 expression is particularly prominent in skeletal, cardiac, and smooth muscle, Leydig cells, endothelia, chondrocytes, and some neurons (3)(4)(5)(6)(7)(8). Like many annexins, Anx2 and Anx5 are predominantly intracellular, existing both as soluble cytosolic proteins and also in Ca 2ϩ -dependent and Ca 2ϩ -independent association with a range of intracellular membranes (9)(10)(11)(12), including the plasma membrane and early endosomes (13,14). However, only Anx5 has been associated with late endosomes and mitochondria (15).…”