Characterisation of the major intrinsic protein (MIP) from bovine lens fibre membranes by electron microscopy and hydrodynamics¹

Abstract

The major intrinsic protein (MIP) from bovine lens fibre membranes has been purified from unstripped membranes using a single ion-exchange chromatography step (MonoS) in the non-ionic detergent octyl-β-d-glucopyranoside (OG). SDS-PAGE has confirmed the purity of the preparation and thin-layer chromatographic analysis has shown that the protein is virtually lipid-free. To establish a stable and monodisperse protein sample, we exchanged OG with decyl-β-d-maltopyranoside (DeM), another non-ionic detergent, by gel-filtration column chromatography. We conclude that the resulting protein/detergent complex is composed of four copies of MIP (a tetramer) and a detergent micelle. This conclusion is based on: (1) measurement of the weight-average molecular mass ($\text{M}$_w,app) of the protein moiety in the protein/detergent complex by sedimentation equilibrium; (2) measurement of the apparent molecular mass of the complexes formed by MIP in OG, in DeM, in dodecyl-β- d-maltopyranoside (DoM) and in sodium dodecylsulphate (SDS) by gel filtration; (3) measurement of the apparent molecular mass of pure detergent micelles; (4) measurement of the predicted change in the molecular mass of the MIP/DeM complex after partial enzymatic proteolysis; and (5) measurement of the size and shape of the MIP/detergent complex by electron microscopy and single-particle analysis. Therefore, the tetragonal arrangement of MIP observed in both plasma membranes and junctional membranes in lens fibre cells is maintained in solution with non-ionic detergents.

Introduction

The vertebrate eye lens is composed of two cell types, an anterior layer of metabolically active epithelial cells and a differentiated cell type, the fibre cells, comprising the bulk of the lens. Differentiation of fibre cells involves a progressive loss of organelles, an accumulation of soluble crystallines in the cytoplasm, together with a substantive increase in plasma membrane as the fibre cells elongate. This formation of new plasma membrane is accompanied by changes in its biochemical composition; the most prominent one is the appearance of increased amounts of a membrane protein with an apparent molecular mass of 28 kDa, called the major intrinsic protein (MIP; Broekhuyse et al., 1976). MIP represents in the mature fibre cell greater than 50% of the total membrane protein and is considered to be a lens differentiation marker (Vermorken et al., 1977).

Cloned and sequenced in 1984, MIP did not show any homology to known membrane proteins (Gorin et al., 1984). In following years, several cDNAs were cloned from mammals, plants, yeast, Drosophila and bacteria that shared up to ∼40% sequence identity with MIP. Proteins of this new emerging gene family, the MIP family Chepelinsky 1994, Pao et al 1991, Reizer et al 1993, share a common membrane topology composed of six putative transmembrane domains with N and C termini exposed on the cytoplasmic surface.

Several studies, using immunocytochemical techniques, have shown that MIP is localised in both lens plasma membranes and lens junctions. These junctions, measuring 11 to 13 nm in overall thickness, contain tetragonal crystals of MIP exhibiting a characteristic spacing of ∼7 nm Costello et al 1989, Dunia et al 1985, Paul and Goodenough 1983 in one membrane abutted against a protein-free apposing membrane (Zampighi et al., 1989). Such a structure is completely different from that of the “gap junctions”, where the plasma membranes of the two adjacent cells both contain arrays of channels that join across the intercellular gap to form channels connecting the two cells, thus providing direct cell-to-cell communication. In addition, MIP exhibits no amino acid sequence homology to the gap junction protein family (Beyer et al., 1990). Therefore, MIP does not mediate direct cell-to-cell communication in the lens.

In an attempt to understand better the structure and function of MIP, the protein has been studied in vivo, in detergent solutions and in artificial membranes. Reconstitution into liposomes and planar lipid bilayers has focused on the channel properties of MIP Ehring et al 1990, Kushmerick et al 1995, Nikaido and Rosenberg 1985, Peracchia et al 1985, Zampighi et al 1985 and on its possible function in membrane adhesion (Michea et al., 1994). In support of the hypothesis of a channel function for MIP in the lens, other homologous members of the MIP family have been shown to form water channels in mammalian tissues, e.g. CHIP28 (Preston et al., 1992), and various specialised channels in prokaryotes and eukaryotes.

It has been noted that the in vivo arrangement into tetragonal arrays, together with their 7 nm spacing, requires that the 28 kDa MIP protein should form aggregates, probably tetramers Ehring et al 1990, Peracchia and Peracchia 1980. When MIP is reconstituted into liposomes and visualised by freeze-fracture, it appears as annular shapes, with diameters of either 6 to 7 nm (Ehring et al., 1990), ∼8 nm Dunia et al 1987, Peracchia et al 1985 or ∼10 nm (Zampighi et al., 1985). An approximately 7 nm particle would accommodate a symmetrical tetramer, as shown in the recent projection structures of the homologous, similarly sized CHIP28 Jap and Li 1995, Mitra et al 1995, Mitra et al 1994, Walz et al 1995.

Studies of detergent-solubilized and purified MIP have focused on the characterisation of the oligomeric state of the protein. Several MIP isolation procedures have used the non-ionic, non-denaturing detergent octyl-β-d-glucopyranoside (OG) to solubilize urea, alkali or guanidine hydrochloride-pretreated lens membranes (stripped membranes). Biophysical characterisation of the MIP/OG complexes, involving calculations from the sedimentation coefficient, have led to contradictory results with respect to the oligomeric state of MIP and concluded MIP in OG to be substantially monomeric (Manenti et al., 1988) or tetrameric (Aerts et al., 1990).

To resolve this controversy and facilitate further functional and structural work on MIP, we sought to develop a new purification method to produce a pure, homogeneous and stable preparation. Cation-exchange chromatography in OG proved to be the single, highly efficient purification step, which in addition allowed us to use unstripped fibre cell membranes. The choice of the appropriate final detergent (exchange of OG for decyl-β-d-maltopyranoside (DeM)) was crucial in stabilising a monodisperse preparation.

Characterisation of the purified, delipidated MIP-DeM preparation, using gel-filtration chromatography, equilibrium ultra centrifugation, electron microscopy and single-particle analysis methods, showed that MIP in DeM is a monodisperse tetramer, exhibiting a side length of ∼8 nm with 4-fold symmetry and a thickness normal to the membrane plane of ∼4 nm.