Optical recording of signal-mediated protein transport through single nuclear pore complexes - PubMed (original) (raw)

Optical recording of signal-mediated protein transport through single nuclear pore complexes

O Keminer et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Optical single-transporter recording, a recently established fluorescence microscopic method, was used to study the selective transport of proteins through single nuclear pore complexes (NPCs) of Xenopus oocytes. Recombinant proteins containing either a nuclear localization signal (import protein) or a nuclear export signal (export protein) were generated as transport substrates. To approximate in vivo conditions as closely as possible, a Xenopus egg extract was applied to the cytosolic side and a Xenopus oocyte nuclear extract to the nuclear side of the NPCs. It was found that protein transport through functionally isolated, "patched" NPCs depended on signal sequences, extracts, and metabolic energy, as in vivo. All NPCs were competent for both import and export. The transport direction was strictly determined by the transport signal, and at none of the conditions explored was the import protein exported or the export protein imported, even when the application sides of the extracts were reversed. The mean transport rates of the single NPC were approximately 2 dimers/s for the import protein and approximately 4 dimers/s for the export protein ( approximately 15 microM substrate concentration, 22-24 degrees C), in good agreement with in vivo rates estimated for mammalian cells by microinjection experiments. The study shows that optical single-transporter recording permits the analysis of membrane transport processes not previously accessible to single-transporter recording and thus provides additional possibilities for the elucidation of nucleocytoplasmic transport mechanisms.

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Figures

Figure 1

Figure 1

Optical recording of signal-mediated protein transport through single NPCs. (A) The principle. The nuclear envelope of Xenopus oocytes is firmly attached to isoporous filters. Filter pores are sealed by mineral oil. Transport into or out of individual filter pores is measured by confocal laser scanning microscopy. (B) The transport substrates. The recombinant green fluorescent proteins IP, EP, and CP contained either a nuclear localization signal (NLS), nuclear export signal (NES), or no localization signal. (C) Import of IP. Filter pores were filled with a solution containing IP (green fluorescence), TRD70 (red fluorescence), and egg extract while a nuclear extract was applied to the unclear side. Upon addition of ATP (0 min) IP was transported out of the filter pores via the NPCs. Simultaneously, the constant TRD70 fluorescence indicated the tightness of sealing and the integrity of the NPCs. (D) Export no EP. EP was added to nuclear side and IP was omitted on the cytoplasmic side, otherwise analogous to C.

Figure 2

Figure 2

Import of IP and export of EP depend on signal sequences, extracts providing soluble transport cofactors, and metabolic energy, and can be inhibited by WGA. (A) From confocal scans as shown in Fig. 1_C_ the time-dependent fluorescence of many filter pores was derived and averaged. Omission of extracts, addition of the ATPase apyrase, or the lectin WGA inhibits transport. (B) Analogous experiments pertaining to the export of EP. (C) CP, containing no localization signal, is neither imported nor exported.

Figure 3

Figure 3

Directionality of transport. The application of the recombinant green fluorescent proteins IP and EP and of egg and nuclear extract to the cytoplasmic and nuclear side of the NPC was permutated to obtain clues for the parameters determining the directionality of transport. See text for details.

Figure 4

Figure 4

Protein transport rate of the NPC. (A and B) From confocal scans as shown in Fig. 1 C and D the time-dependent fluorescence of single-filter pores was derived. Fluorescence was converted into concentration and fitted by a simple exponential. The fit was used to derive the initial rate and, taking the volume of the filter pore into account, expressed in molecules/s per patch. (C_–_F) Populations of transport rates were plotted as frequency diagrams where C and D are the raw data, and E and F are smoothed data. The peaks that are equidistantly spaced on the abscissa represent membrane patches with one, two, or three NPCs.

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