Direct evidence for the transmembrane orientation of the hepatic glycoprotein receptors.

127

148

Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

Section: Analysis Of Reporter Proteins With An Enlarged Cytosolic or Lumenal Domainmentioning

confidence: 99%

Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

Ohba

Schirmer

Nishimoto

et al. 2004

127

148

“…Chicken LBR is an integral protein of the inner nuclear m e m b r a n e with a basic, nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic, carboxyl-terminal domain of 433 amino acids with eight putative transmembrane segments (Worman et al, 1990). CHL is a type II integral m e m b r a n e protein localized to the ER, endosomes, and plasma m e m b r a n e that has an amino-terminal domain of 23 amino acids that faces the cytoplasm, a single transmembrane segment and a luminal, carboxylterminal domain of 160 amino acids (Chiacchia and Drickamer, 1984;Mellow et al, 1988). The truncated version of CMPK is a completely soluble, normally cytosolic protein of ~5 0 kD (Frangioni and Neel, 1993).…”

Section: Targeting Studiesmentioning

confidence: 99%

Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane.

Soullam

Worman

1995

202

180

Abstract. We have examined transfected cells by immunofluorescence microscopy to determine the signals and structural features required for the targeting of integral membrane proteins to the inner nuclear membrane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nucleoplasmic, amino-terminal domain and a carboxylterminal domain with eight putative transmembrane segments. The amino-terminal domain of LBR can target both a cytosolic protein to the nucleus and a type II integral protein to the inner nuclear membrane. Neither a nuclear localization signal (NLS) of a soluble protein, nor full-length histone H1, can target an integral protein to the inner nuclear membrane although they can target cytosolic proteins to the nucleus. The addition of an NLS to a protein normally located in the inner nuclear membrane, however, does not inhibit its targeting. When the amino-terminal domain of LBR is increased in size from ~22.5 to ,'~70 kD, the chimeric protein cannot reach the inner nuclear membrane. The carboxyl-terminal domain of LBR, separated from the amino-terminal domain, also concentrates in the inner nuclear membrane, demonstrating two nonoverlapping targeting signals in this protein. Signals and structural features required for the inner nuclear membrane targeting of proteins are distinct from those involved in targeting soluble polypeptides to the nucleoplasm. The structure of the nucleocytoplasmic domain of an inner nuclear membrane protein also influences targeting, possibly because of size constraints dictated by the lateral channels of the nuclear pore complexes.

“…To test this hypothesis, we exchanged the classical signal sequence of LT-␣ for the membrane anchor structure of CHL (Mellow et al, 1988); by choosing the latter instead of the TNF presequence we avoided any possible specific functional contribution of the TNF presequence. CHL is a trimeric, type II transmembrane liver glycoprotein (Chiacchia et al, 1984;Steer et al, 1990), which contains a membrane anchor allowing trimerization of the extracellular domain. Transfection of L929r2 cells with the neo r gene, combined with the CHL.hLT-␣ chimeric gene (Fig.…”

Section: Introduction Of a Membrane-anchoring Step In The Biosynthesis Pathway Of Hlt-␣ Induces Tnf/lt-␣ Resistance And Downmodulation Ofmentioning

confidence: 99%

Autocrine Tumor Necrosis Factor (TNF) and Lymphotoxin (LT) α Differentially Modulate Cellular Sensitivity to TNF/LT-α Cytotoxicity in L929 Cells

Decoster

Cornelis

Vanhaesebroeck

et al. 1998

Tumor necrosis factor (TNF) and lymphotoxin (LT) α are structurally and functionally related cytokines. We expressed the TNF and LT-α genes in murine fibrosarcoma L929r2 cells, which can be sensitized to TNF/LT-α–dependent necrosis by inhibitors of transcription or translation. Autocrine production of murine TNF in L929r2 cells completely downmodulated the expression of the 55- and 75-kD TNF receptors, resulting in resistance to TNF/LT-α cytotoxicity. Partial downmodulation of the 55-kD receptor was observed in human TNF-producing L929r2 cells. In contrast, an unaltered TNF receptor expression was found on LT-α L929r2 transfectants. Hence, although similar cytotoxic effects are induced by extracellularly administered TNF and LT-α, endogenous expression of these cytokines fundamentally differs in the way they modulate TNF receptor expression. Unlike LT-α, secreted by the classical pathway, TNF is first formed as a membrane-bound protein, which is responsible for receptor downmodulation. To explore whether the different pathways for secretion of TNF and LT-α explain this difference, we examined the effect of membrane-bound LT-α expression. This was obtained by exchange of the classical signal sequence of LT-α for the membrane anchor of chicken hepatic lectin. Membrane retention of LT-α resulted indeed in receptor downmodulation and TNF/LT-α resistance. We conclude that membrane retention of newly synthesized TNF or LT-α is absolutely required for receptor downmodulation and TNF/LT-α resistance.