In vivo dynamics of nuclear pore complexes in yeast

被引:92
作者
Bucci, M [1 ]
Wente, SR [1 ]
机构
[1] WASHINGTON UNIV, SCH MED, DEPT CELL BIOL & PHYSIOL, ST LOUIS, MO 63110 USA
关键词
D O I
10.1083/jcb.136.6.1185
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea ea victoria and expressed in nup49 null haploid yeast cells. When the GFP-Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process, By monitoring the distribution of the GFP-Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus, We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion, When assays were done in mutant karl-l strains, where nuclear fusion does not occur, GFP-Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP-Nup49p into existing recipient NPCs, Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures, This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point, Using the GFP-Nup49p system with a mutant in the NPC-associated factor Gle2p that exhibits formation of NPC clusters only at 37 degrees C, it was possible to distinguish between these two models for NPC dynamics. GFP-Nup49p-labeled NPCs, assembled at 23 degrees C, moved into clusters when the cells were shifted to growth at 37 degrees C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains, Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation, This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.
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页码:1185 / 1199
页数:15
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共 75 条
[1]   Nup120p: A yeast nucleoporin required for NPC distribution and mRNA transport [J].
Aitchison, JD ;
Blobel, G ;
Rout, MP .
JOURNAL OF CELL BIOLOGY, 1995, 131 (06) :1659-1675
[2]   2 NOVEL RELATED YEAST NUCLEOPORINS NUP170P AND NUP157P - COMPLEMENTATION WITH THE VERTEBRATE HOMOLOG NUP155P AND FUNCTIONAL INTERACTIONS WITH THE YEAST NUCLEAR PORE-MEMBRANE PROTEIN POM152P [J].
AITCHISON, JD ;
ROUT, MP ;
MARELLI, M ;
BLOBEL, G ;
WOZNIAK, RW .
JOURNAL OF CELL BIOLOGY, 1995, 131 (05) :1133-1148
[3]   STRUCTURAL PLASTICITY OF THE NUCLEAR-PORE COMPLEX [J].
AKEY, CW .
JOURNAL OF MOLECULAR BIOLOGY, 1995, 248 (02) :273-293
[4]   YEAST NUCLEAR-ENVELOPE PROTEINS CROSS REACT WITH AN ANTIBODY AGAINST MAMMALIAN PORE COMPLEX PROTEINS [J].
ARIS, JP ;
BLOBEL, G .
JOURNAL OF CELL BIOLOGY, 1989, 108 (06) :2059-2067
[5]   A SIMPLE AND EFFICIENT METHOD FOR DIRECT GENE DELETION IN SACCHAROMYCES-CEREVISIAE [J].
BAUDIN, A ;
OZIERKALOGEROPOULOS, O ;
DENOUEL, A ;
LACROUTE, F ;
CULLIN, C .
NUCLEIC ACIDS RESEARCH, 1993, 21 (14) :3329-3330
[6]   GTP HYDROLYSIS IS REQUIRED FOR VESICLE FUSION DURING NUCLEAR-ENVELOPE ASSEMBLY INVITRO [J].
BOMAN, AL ;
DELANNOY, MR ;
WILSON, KL .
JOURNAL OF CELL BIOLOGY, 1992, 116 (02) :281-294
[7]   A ROLE FOR ADP-RIBOSYLATION FACTOR IN NUCLEAR VESICLE DYNAMICS [J].
BOMAN, AL ;
TAYLOR, TC ;
MELANCON, P ;
WILSON, KL .
NATURE, 1992, 358 (6386) :512-514
[8]   MULTIFUNCTIONAL YEAST HIGH-COPY-NUMBER SHUTTLE VECTORS [J].
CHRISTIANSON, TW ;
SIKORSKI, RS ;
DANTE, M ;
SHERO, JH ;
HIETER, P .
GENE, 1992, 110 (01) :119-122
[9]   MUTANT OF SACCHAROMYCES-CEREVISIAE DEFECTIVE FOR NUCLEAR FUSION [J].
CONDE, J ;
FINK, GR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1976, 73 (10) :3651-3655
[10]   IDENTIFICATION OF A SOLUBLE PRECURSOR COMPLEX ESSENTIAL FOR NUCLEAR-PORE ASSEMBLY INVITRO [J].
DABAUVALLE, MC ;
LOOS, K ;
SCHEER, U .
CHROMOSOMA, 1990, 100 (01) :56-66