The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope.

Abstract

Abstract. The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored, gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOHterminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092 Greber, U. E, A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components.T hE nuclear envelope (NE) ~ consists of three morphologically and biochemically distinct domains (for review see 9, 12). The outer nuclear membrane domain contains bound ribosomes and is continuous with and biochemically indistinguishable from the RER. The inner nuclear membrane domain is adjacent to the nuclear lamina and the underlying chromatin, and contains a distinct set of integral membrane proteins (1,29,36). Finally, the outer and inner membranes of the NE are connected with each other at numerous sites forming circular openings in the NE (nuclear pores) of "~100 nm diam. The connecting membrane is sharply bent (180~ and is referred to as the "pore membrane" domain of the NE. The nuclear pore is occupied by the nuclear pore complex (NPC) and the pore membrane is adjacent to the NPC (for review see 9, 21). The pore membrane contains at least one distinct glycoprotein, referred to as gp210 (10, 37). This, and perhaps other yet to be identified pore membrane-specific integral proteins, may function in nuclear pore formation and in the assembly and attachment of the NPC to the pore membrane.Gp210 has been estimated to be present in 16-24 copies per nuclear pore (9). Its primary structure has been deduced from overlapping cDNA clones (37), and its topology has been determined using proteolytic enzymes and domainspecific antibodies (11; E. Bartnik, R. W. Wozniak, and G.1. Abbreviations used in this paper: CT, cytoplasmic tail; HA, hemagglutinin; NE, nuclear envelope; NPC, nuclear pore complex; PCR, polymerase chain reaction; TM, transmembrane segment.Blobel, unpublished results), gp210 has an estimated mass of...

The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope

“…Our data suggest that the coiled-coil region of Sun1 is important to mediate NE association, however the ability of the Sun1-TM-CΔCC fusion protein to localise to the NE is indicative of the existence of additional nuclear retention signal(s) in the C- terminus of Sun1. Furthermore we cannot exclude the possibility that the Sun1 transmembrane domains themselves contain sorting signals and determine the NE localisation in a similar fashion to the lamin B receptor (Wozniak and Blobel, 1992;Smith and Blobel, 1993). The presence of multiple and independent nuclear retention signals across Sun1 is further supported by the fact that none of the fusion proteins localised as efficiently to the NE as the full-length Sun1 (Fig.…”

Topology and Functional Domains of the Yeast Pore Membrane Protein Pom152p

“…In this regard, it is similar to the mammalian pore membrane protein gp210 (5,8), which has 95% of its mass positioned on the lumenal side of the membrane. This region of gp210 likely contributes to lumenal structures such as the lumenal spokes or radial arms, and it has been proposed that this region plays a role in pore formation and the maintenance of NPC structure (5,8,10). Moreover, antibodies to the lumenal domain of gp210 partially inhibit nuclear import of classical nuclear localization signalcontaining substrates (25).…”

“…It has been proposed that after their integration into the endoplasmic reticulum membrane, these proteins laterally diffuse through the lipid bilayer to their destination within the pore membrane (10). For gp210, the transmembrane segment is the dominant sorting determinant for its targeting to the pore membrane (10).…”

Nuclear translocation of mutagenized forms of human cytomegalovirus glycoprotein B (gpUL55).

“…On the other hand, much less is known about the mode of translocation of transmembrane proteins into the inner nuclear membrane compartment (INM). So far, targeting to the INM of the lamin B receptor (LBR ; Smith & Blobel, 1993 ;Soullam & Worman, 1993), the nuclear pore complex protein gp210 (Wozniak & Blobel, 1992) and herpes simplex virus type 1 glycoprotein B (HSV-1 gB ; Raviprakash et al, 1990 ;Rasile et al, 1993 ;Gilbert et al, 1994) has been investigated. In Author for correspondence : K. Radsak.…”