An integral membrane protein from the nuclear pore complex is also present in the annulate lamellae: implications for annulate lamella formation.
Annulate lamellae (AL) are cytoplasmic arrays of stacked membrane cisternae containing densely packed pore complexes which are similar in structure to the nuclear pore complexes (NPCs) and thus referred to as annulate lamella pore complexes (ALPCs). We have recently shown that the integral nuclear pore membrane protein POM121 tagged with green fluorescent protein was correctly targeted to the nuclear pores (H. Söderqvist et al., 1997, Eur. J. Biochem. 250, 808-813). Here we have investigated if POM121 fused to three tandem molecules of yellow fluorescent protein (YFP) (POM121-YFP(3)) also was able to distribute in the extensive and well-characterized AL of RC37 and BMGE cells. Transfected RC37 or BMGE cells displayed YFP fluorescence around the nuclear envelope, as well as in the cytoplasmic AL structures. The YFP fluorescence colocalized perfectly with immunostaining using antibodies specific for different NPC proteins. The AL of both transfected and untransfected BMGE cells resisted extractions with Tx-100 and 250 mM NaCl, but were completely solubilized at 450 mM NaCl. Loss of YFP fluorescence and immunostaining for other NPC proteins correlated under all extraction conditions tested, suggesting that overexpressed POM121-YFP(3) had become an integrated part both of the NPCs and of the ALPCs. Furthermore, we have generated a stable BHK cell line expressing POM121-YFP(3) located exclusively at the nuclear pores. Treatment with vinblastine sulfate, which induces formation of AL in a variety of cells, resulted in distribution of POM121-YFP(3) into cytoplasmic foci colocalizing with immunostaining for peripheral NPC proteins. Taken together, the results show that YFP-tagged POM121 is able to distribute in drug-induced or naturally occurring AL, suggesting that POM121 is a natural constituent of ALPCs. In COS cells, which normally lack or have very little AL, YFP-tagged POM121 distributed in the nuclear pores when expressed at low levels. However, at high expression levels the YFP fluorescence also distributed in a number of brightly fluorescing cytoplasmic dots or foci, which were not present in untransfected cells. This was also true for untagged POM121. The cytoplasmic foci varied in size from 0. 1 to 2 microm and were distinctly located in the immediate vicinity of ER cisternae (without colocalizing) and also contained other nuclear pore proteins, indicating that they may represent cytoplasmic AL. This idea is supported by time-lapse studies of postmitotic assembly of these structures. This raises the question of the role of POM121 in ALPC and NPC biogenesis.
Imreh
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G
Södertörns Högskola (University Collage), Huddinge, S-141 04, Sweden.
Hallberg
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D000818:Animals; D000906:Antibodies; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D019556:COS Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D009685:Nuclear Envelope / Q000737:chemistry* / Q000502:physiology*; D009687:Nuclear Proteins; D010957:Plasmids; D014162:Transfection; D014747:Vinblastine / Q000494:pharmacology
D000906:Antibodies; D000972:Antineoplastic Agents, Phytogenic; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; C082264:Pom121 protein, rat; D049452:Green Fluorescent Proteins; D014747:Vinblastine