In this issue of Blood, Satchwell and colleagues provide compelling evidence that during the genesis of the plasma membrane in differentiating erythroblasts, key components of band 3/Rh multiprotein complexes can traffic to the membrane already linked or assemble together for the first time in the membrane.1 

During erythropoiesis the plasma membrane is in a dynamic state of organization as newly synthesized proteins incorporate into the lipid bilayer. Throughout terminal differentiation the erythroblast membrane facilitates key cellular properties including adhesive interactions with macrophages, active endocytosis of iron, a progressive decrease in cell volume and increased hemoglobin synthesis. Although the timing of expression of individual proteins on the plasma membrane has been described, little is known about the assembly of these individual components into large multiprotein complexes that are known to be present in the mature erythrocyte membrane. Among the important physiologic roles of one of these multiprotein complexes are anion, NH3 and CO2 transport and maintenance of membrane cohesion.2  Major questions still to be answered include: At what point in erythropoiesis do these complexes assemble? And are these complexes assembled after incorporation of individual components in the membrane or are they assembled intracellularly and then trafficked to the membrane?

The most abundant multiprotein complex is the ankyrin-based band 3 macrocomplex, impressive in size, and containing both a band 3 subcomplex and a Rh subcomplex.3  The band 3 subcomplex includes the erythrocyte anion transporter band 3, which associates with glycophorin A and links to the cytoskeleton via protein 4.2 and ankyrin. These interactions are important for membrane cohesion and their absence in hereditary spherocytosis (HS) leads to surface area loss.4  The NH3 and CO2 transporter RhAG5,6  forms a heterotetramer with RhCe and RhD (termed Rh polypeptides), and this core complex interacts with proteins CD47, LW, and glycophorin B, together forming the Rh subcomplex. These linked proteins are deficient in the Rh null phenotype leading to hemolytic anemia with stomatocytes and spherocytes because of loss of surface area.7 

To begin to explore the spatio-temporal mechanisms of the band 3 macrocomplex formation during erythropoiesis, Satchwell and colleagues focused their studies on 2 of the crucial linkages within the macrocomplex, band 3/protein 4.2 and RhAG/Rh. Using erythroid cultures from human peripheral blood mononuclear cells, the authors applied confocal immunofluorescent microscopy and biochemical techniques to study the intracellular trafficking of these proteins and the dynamics of their interactions. They observed that the bulk of band 3 and RhAG is delivered to the plasma membrane early during differentiation. Moreover, in basophilic erythroblast plasma membranes, band 3 and protein 4.2 are already present in a linked state. Interestingly, they found band 3 associating with protein 4.2 in the early stages of the secretory pathway, either in the early Golgi or ER, providing evidence that the 2 newly synthesized proteins traffic together to the membrane. In contrast, no intracellular interaction of RhAG and Rh was detected. However the 2 polypeptides could be coimmunoprecipitated from plasma membranes of basophilic erythroblasts, signifying that they link within the membrane after their individual incorporation. In addition, knockdown of RhAG during early erythropoiesis resulted in decreased expression of Rh polypeptides, indicating that the stability of Rh in the membrane requires the presence of RhAG. Together these findings establish for the first time that major, core components of the band 3 macrocomplex have assembled in early-stage erythroblasts.

These data generate a host of intriguing questions of significance to both erythroid biology and clinical medicine. In particular, what are the anion, NH3 and CO2 transport capabilities of partially assembled complexes compared with fully assembled complexes? And in a related issue, at what point during differentiation do these ion and gas transport apparatuses need to be functional for the health of the cell? Another question is at what stage of differentiation do the membrane complexes associate with the cell cytoskeleton? In erythrocytes, vertical interactions between the lipid bilayer and the cytoskeleton created by these multiprotein complexes are crucial for maintaining membrane cohesion and protecting cells from surface area loss.2  One might postulate that when exuberant endocytosis of iron is required for hemoglobin synthesis, a less cohesive membrane might be advantageous to facilitate endocytosis. Thus future studies may show that lipid bilayer/cytoskeleton linkages mediated by the band 3 macrocomplex form at a late stage of differentiation. A third issue to consider has therapeutic implications for patients with HS. In HS, a mutation in a gene encoding one component of the band 3 complex (ankyrin, band 3 or protein 4.2), leads to a decrease in the other components of the complex in mature erythrocytes. One mechanism known to generate these deficiencies is their loss during the process of erythroblast protein partitioning to the reticulocyte membrane at the time of enucleation.8  However it is also possible that the band 3 complex does not assemble normally during erythropoiesis, leading to ineffective erythropoiesis. This would be a novel and as yet unexplored mechanism contributing to the anemia of HS, which until now has been considered solely because of hemolysis. If ineffective erythropoiesis is, indeed, an important contributor to the anemia in some cases of HS, it would have therapeutic implications, because splenectomy might not successfully ameliorate the severity of anemia.

Conflict-of-interest disclosure: The author declares no competing financial interests. ■

1
Satchwell
 
TJ
Bell
 
AJ
Pellegrin
 
S
, et al. 
Critical band 3 multiprotein complex interactions establish early during human erythropoiesis.
Blood
2011
, vol. 
118
 
1
(pg. 
182
-
191
)
2
Mohandas
 
N
Gallagher
 
PG
Red cell membrane: past, present, and future.
Blood
2008
, vol. 
112
 
10
(pg. 
3939
-
3948
)
3
Bruce
 
LJ
Beckmann
 
R
Ribeiro
 
ML
, et al. 
A band 3-based macrocomplex of integral and peripheral proteins in the RBC membrane.
Blood
2003
, vol. 
101
 
10
(pg. 
4180
-
4188
)
4
Peters
 
LL
Shivdasani
 
RA
Liu
 
SC
, et al. 
Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton.
Cell
1996
, vol. 
86
 
6
(pg. 
917
-
927
)
5
Ripoche
 
P
Bertrand
 
O
Gane
 
P
Birkenmeier
 
C
Colin
 
Y
Cartron
 
JP
Human Rhesus-associated glycoprotein mediates facilitated transport of NH(3) into red blood cells.
Proc Natl Acad Sci U S A
2004
, vol. 
101
 
49
(pg. 
17222
-
17227
)
6
Endeward
 
V
Carton
 
JP
Ripoche
 
P
Gros
 
G
RhAG protein of the Rhesus complex is a CO2 channel in the human red cell membrane.
FASEB J
2008
, vol. 
22
 
1
(pg. 
64
-
73
)
7
van den Akker
 
E
Satchwell
 
TJ
Williamson
 
RC
Toye
 
AM
Band 3 multiprotein complexes in the red cell membrane; of mice and men.
Blood Cells Mol Dis
2010
, vol. 
45
 
1
(pg. 
1
-
8
)
8
Salomao
 
M
Chen
 
K
Villalobos
 
J
Mohandas
 
N
An
 
X
Chasis
 
JA
Hereditary spherocytosis and hereditary elliptocytosis: aberrant protein sorting during erythroblast enucleation.
Blood
2010
, vol. 
116
 
2
(pg. 
267
-
269
)

National Institutes of Health