THE IDENTIFICATION AND CHARACTERIZATION OF AN INNER ACROSOMAL MEMBRANE ASSOCIATED PROTEIN, IAM38, RESPONSIBLE FOR SECONDARY SPERM-ZONA BINDING DURING FERTILIZATION

During mammalian fertilization, the exposure of the inner acrosomal membrane (IAM) after acrosomal exocytosis is essential for the secondary binding between sperm and zona pellucida (ZP) of the oocyte, a prerequisite for sperm penetration through the ZP. The identification of the sperm protein(s) responsible for secondary binding has posed a challenge for researchers. We were able to isolate a sperm head fraction in which the IAM was exposed. Attached to the IAM was an electon dense layer, which we termed the IAM extracellular coat (IAMC). The IAMC was also observable in acrosome reacted sperm. High salt extraction removed the IAMC including a prominent 38 kDa polypeptide, referred to as IAM38. Antibodies raised against IAM38 confirmed its presence in the IAMC of intact, sonicated, and acrosome-reacted sperm. Sequencing of IAM38 revealed it as the ortholog of porcine SP38, a protein that was found to bind specifically to ZP2 but whose intra-acrosomal location was not known. We showed that IAM38 occupied the leading edge of sperm contact with the zona pellucida during fertilization, and that secondary binding and fertilization were inhibited in vitro by antibodies directed against IAM38. As for the mechanism of secondary sperm-zona binding by IAM38, we provided evidence that the synthetic peptide derived from the ZP2-binding motif of IAM38 had a competitive inhibitory effect on both sperm-zona binding and fertilization while its mutant form was ineffective. In summary, our study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM and consolidates IAM38 as a genuine secondary sperm-zona binding protein. In addition, our investigation also provides an ultrastructural description of the origin, expression and assembly of IAM38 during spermatogenesis. It shows that IAM38 is originally secreted by the Golgi apparatus as part of the dense contents of the proacrosomic granules but later, during acrosome capping phase of spermiogenesis, is redistributed to the inner periphery of the acrosomal membrane. This relocation occurs at the time of acrosomal compaction, an obligatory structural change that fails to occur in Zpbp1-/- knockout mice, which do not express IAM38 and are infertile.

Novel interaction between RET and membrane cytoskeletal-linker protein ezrin

RET is a receptor tyrosine kinase that mediates key signaling events, and promotes cell survival, development, and migration. Activation of RET requires a ligand from the glial cell line-derived neurotrophic factor (GDNF) family and a co-receptor from the GDNF family receptor α (GFRα). Alternative splicing of RET leads to two major isoforms, RET9 and RET51, that contain distinct C-terminal amino acids. Differences in their cytoplasmic tails confer differential binding to adaptor proteins, and in this study, the membrane cytoskeletal-linker protein ezrin was shown in an interaction with RET51, but not RET9, in a ligand- and kinase-dependent manner. Results indicated that Y1096 on RET51 is the ezrin recruitment site, and the adaptor protein Grb2 may mediate this interaction. These results suggest that ezrin may play a role in the downstream signaling and recycling pathways of RET51. Thus, the identified novel interaction may provide insight in the longer term into how ezrin and RET51 contribute together to functional processes such as cell migration and invasion.

Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane

The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKB alpha termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKB alpha at the plasma membrane. Binding of CTMP reduces the activity of PKB alpha by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt-transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

Multiplex analysis of age-related protein and lipid modifications in human Bruch's membrane

Aging of the human retina is characterized by progressive pathology, which can lead to vision loss. This progression is believed to involve reactive metabolic intermediates reacting with constituents of Bruch's membrane, significantly altering its physiochemical nature and function. We aimed to replace a myriad of techniques following these changes with one, Raman spectroscopy. We used multiplexed Raman spectroscopy to analyze the age-related changes in 7 proteins, 3 lipids, and 8 advanced glycation/lipoxidation endproducts (AGEs/ALEs) in 63 postmortem human donors. We provided an important database for Raman spectra from a broad range of AGEs and ALEs, each with a characteristic fingerprint. Many of these adducts were shown for the first time in human Bruch's membrane and are significantly associated with aging. The study also introduced the previously unreported up-regulation of heme during aging of Bruch's membrane, which is associated with AGE/ALE formation. Selection of donors ranged from ages 32 to 92 yr. We demonstrated that Raman spectroscopy can identify and quantify age-related changes in a single nondestructive measurement, with potential to measure age-related changes in vivo. We present the first directly recorded evidence of the key role of heme in AGE/ALE formation.

BcsK(C) Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsL(B)

The type VI secretion system (T6SS) contributes to the virulence of Burkholderia cenocepacia, an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. BcsK(C) is a highly conserved protein among the T6SSs in Gram-negative bacteria. Here, we show that BcsK(C) is required for Hcp secretion and cytoskeletal redistribution in macrophages upon bacterial infection. These two phenotypes are associated with a functional T6SS in B. cenocepacia. Experiments employing a bacterial two-hybrid system and pulldown assays demonstrated that BcsK(C) interacts with BcsL(B), another conserved T6SS component. Internal deletions within BcsK(C) revealed that its N-terminal domain is necessary and sufficient for interaction with BcsL(B). Fractionation experiments showed that BcsK(C) can be in the cytosol or tightly associated with the outer membrane and that BcsK(C) and BcsL(B) form a high molecular weight complex anchored to the outer membrane that requires BcsF(H) (a ClpV homolog) to be assembled. Together, our data show that BcsK(C)/BcsL(B) interaction is essential for the T6SS activity in B. cenocepacia.

Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein

N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway. The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER. The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate. This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion. The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins. The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells. The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive. Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.

The N-terminal region of the Escherichia coli WecA (Rfe) protein, containing three predicted transmembrane helices, is required for function but not for membrane insertion

The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.

Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology

Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein)--haptoEGFPs--which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein-haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.

Purification of a multidrug resistance transporter for crystallization studies

Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.

A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Overproduction, purification and preliminary X-ray diffraction analysis of YncE, an iron-regulated Sec-dependent periplasmic protein from Escherichia coli

The yncE gene of Escherichia coli encodes a predicted periplasmic protein of unknown function. The gene is de-repressed under iron restriction through the action of the global iron regulator Fur. This suggests a role in iron acquisition, which is supported by the presence of the adjacent yncD gene encoding a potential TonB-dependent outer-membrane transporter. Here, the preliminary crystallographic structure of YncE is reported, revealing that it consists of a seven-bladed beta-propeller which resembles the corresponding domain of the `surface-layer protein' of Methanosarcina mazei. A full structure determination is under way in order to provide insight into the function of this protein.