Malini Mansharamani

Direct Binding of Nuclear Membrane Protein MAN1 to Emerin in Vitro and Two Modes of Binding to Barrier-to-Autointegration Factor

MAN1 is a vertebrate nuclear inner membrane protein that inhibits Smad signaling downstream of transforming growth factor β. MAN1 has an exposed LEM domain-containing N-terminal region (“MAN1-N”), two transmembrane domains, and an exposed C-terminal domain (“MAN1-C”). Many regions of human MAN1 are homologous to emerin, a LEM domain nuclear protein, loss of which causes Emery-Dreifuss muscular dystrophy (EDMD). To test the hypothesis that MAN1 function might overlap with emerin, we tested different polypeptide fragments of MAN1 for binding to selected partners of emerin. Our findings support this hypothesis. Blot overlay assays and co-immunoprecipitation studies showed that MAN1-C binds the transcription regulators GCL, Btf, and barrier-to-autointegration factor (BAF). BAF binding to this region, which has no LEM domain, was notable. Sequence alignments identified a potential BAF-binding motif, characterized by the conserved residues Ser-Arg-Val, in MAN1-C and two other BAF-binding proteins. The other region, MAN1-N, bound directly to BAF, lamin A, and lamin B1, supporting functional overlap with emerin. Unexpectedly, three independent assays showed that MAN1-N also bound directly to emerin. Proposed MAN1-emerin complexes are discussed in the context of EDMD disease mechanisms and potential in vivo functions.

The nuclear envelope, lamins and nuclear assembly.

The nuclear lamina is composed of both A- and B-type lamins and lamin-binding proteins. Many lamin-binding proteins are integral proteins of the inner nuclear membrane. Lamins and inner nuclear membrane proteins are important for a variety of cell functions, including nuclear assembly, replication, transcription, and nuclear integrity. Recent advances in the field in the past year include the identification of a family of spectrin-repeat-containing inner nuclear membrane proteins and other novel inner-membrane proteins, and the discovery of a nuclear membrane fusion complex. There is also growing evidence that A- and B-type lamins and their binding partners have distinct roles during nuclear assembly and interphase.

The reproductive importance of P-type ATPases

P-type ATPases are integral membrane proteins that use the free energy of ATP hydrolysis to generate transmembrane electrochemical ion gradients to support a variety of cellular processes. They have eight signature motifs, eight or ten transmembrane domains, highly conserved phosphorylation and ATP-binding sites, and similar hydropathic profiles. This review summarizes recent insights in the relationship of P-type ATPases to successful reproduction, and the hormone dependence of some family members. Because protein topology is central to understanding the pump action of this family of enzymes, this review also describes the dramatic change in the primary structure of one family member that may mediate transcription in the uterus.

Uteroglobin Gene Transcription: What's the RUSH?

Abstract: Prolactin enhances progesterone‐dependent transcription of the rabbit uteroglobin gene. RUSH transcription factors are implicated in the signal transduction pathway. The RUSH acronym identifies key features of these nuclear phosphoproteins, that is, RING‐finger motif, binds the uteroglobin promoter, structurally related to the SWI/SNF family of transcription factors, and helicase‐like. Cloned by recognition site screening, RUSH proteins bind to an 85‐bp region (−170/−85) of the uteroglobin promoter that was subsequently identified as a novel prolactin‐responsive region by promoter deletion analysis. Gel shift and linker‐scanning assays further reduced the RUSH target site to −160/−110. A hexameric core of MCWTDK was identified as the RUSH‐specific DNA‐binding site (−126/−121) by CASTing. This site overlaps authentic HNF3b and OCT‐1 binding sites. A unique Type IV P‐type ATPase that is embedded in the inner nuclear membrane binds the RING domain of RUSH. The conformationally flexible loop portion of this RING‐finger binding protein (RFBP) extends into the nucleoplasm to contact euchromatin. The physical association of RFBP with transcriptionally active chromatin supports the speculation that RFBP targets RUSH transcription factors to the active uteroglobin promoter.

Prolactin Augments Progesterone‐Dependent Expression of a Nuclear P‐Type ATPase that Associates with the RING Domain of RUSH Transcription Factors in the Endometrium

Links between hormone action, gene structure and function, and evolution comprise some of the most compelling themes of molecular endocrinology. Multiple sequence alignments have played a key role in the identification of functionally important regions in gene families. The RING-finger motif is an example of a sequence-structure family1 that is purported to be involved in the formation of large protein complexes.2 Originally identified in only 7 proteins in the OWL database,3 the RING motif is the common feature of a superfamily of nearly 200 otherwise unrelated proteins, many of which are transcription factors. In an effort to understand the function of the RING domain, the search for RING-finger binding partners has become quite intense. We recently cloned and characterized a RING-Finger Binding Protein (RFBP; Genbankuf6db accession number AF236061) from a rabbit uterine epithelial (HRE-H9) cell line. RFBP binds the RING domain of RUSH nuclear phosphoproteins that are potential mediators4 of uteroglobin gene transcription. The coding sequence for the RFBP has all of the structural features that are diagnostic of a Type IV P-type ATPase,5 including seven of eight core segments and nine transmembrane domains potentially involved in substrate binding and translocation. P-type ATPases are classified into five main groups based on substrate specificity and sequence identity.5 These groups include Type I (heavy metal pumps), Type II (Ca2+, Na+/K+, and H+/K+ pumps), Type III (H+ and Mg2+ pumps), Type IV (phospholipid pumps), and Type V (no assigned substrate specificity). A search of sequence databases using the BLAST program at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) revealed that the predicted RFBP protein and Type IV P-type ATPases from Bos taurus (protein ID AAD03352), Mus musculus (protein ID AAB18627), and Saccharomyces cerevisiae (protein ID L01795) are 50% similar and 34% identical. The three Type IV P-type ATPases are putative aminophospholipid transporters. However, the newly identified RFBP differs from these authentic Type IV P-type ATPases in two important ways. The most remarkable feature is that it has an odd number of transmembrane domains. All known P-type ATPases have an even num-

Erratum to “The reproductive importance of P-type ATPases”: [Mol. Cell. Endocrinol. 183 (2001) 123–126]

P-type ATPases are integral membrane proteins that use the free energy of ATP hydrolysis to generate transmembrane electrochemical ion gradients to support a variety of cellular processes. They have eight signature motifs, eight or ten transmembrane domains, highly conserved phosphorylation and ATP-binding sites, and similar hydropathic profiles. This review summarizes recent insights in the relationship of P-type ATPases to successful reproduction, and the hormone dependence of some family members. Because protein topology is central to understanding the pump action of this family of enzymes, this review also describes the dramatic change in the primary structure of one family member that may mediate transcription in the uterus.