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© The Rockefeller University Press, 0021-9525/1999//839 $5.00
The Journal of Cell Biology, Volume 144, Number 5, , 1999 839-855

Regular Articles

Proteins Connecting the Nuclear Pore Complex with the Nuclear Interior



Caterina Strambio-de-Castillia{ddagger}, Günter Blobel*, and Michael P. Rout{ddagger}

* Laboratory of Cell Biology, Howard Hughes Medical Institute; and {ddagger} Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10021

While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC.

Key Words: nucleocytoplasmic transport • nucleoskeleton • nuclear pore complex • nuclear envelope • Saccharomyces cerevisiae

Abbreviations used in this paper: GFP, green fluorescent protein; IEM, immunoelectron microscopy; IF, immunofluorescence; MLP, myosin-like protein; NE, nuclear envelope; NES, nuclear export sequence; NPC, nuclear pore complex; NUP, nucleoporin; PVP, polyvinylpyrrolidone.

Address correspondence to Dr. G. Blobel, Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021. Tel.: (212) 327-8096. Fax: (212) 327-7880. E-mail: blobel{at}rockvax.rockefeller.edu

We are very grateful to J. Aitchison, C. Akey, R. Beckmann, N. Bonifaci, Y. Chook, E. Coutavas, U. O'Doherty, R. Erdmann, B. Fontoura, J. Helmers, M. Hurwitz, E. Johnson, J. Kilmartin, M. Matunis, L. Pemberton, and S. Smith for many helpful suggestions and discussions throughout the course of this study. We are deeply indebted to J. Aris, C. Cole, D. Goldfarb, E. Johnson, J. Kilmartin, M. Lewis, L. Pemberton, M. Sogaard, K. Weis, and R. Wozniak, for providing us with antibodies and other reagents without which this work would not have been possible and to T. De Lange and J. Karlsreder for assistance in collecting the immunofluorescence data presented here. Many thanks also to R. Beckmann, Y. Chook, and J. Luban for critical reading of the manuscript in whole or in part. Special thanks go to H. Shio and E. Sphicas for excellent technical assistance in the electron microscopic studies. Our sincerest gratitude goes to E. Ellison, H. Ijikata, and Y. Oh for invaluable and skillful technical support that was essential for the completion of various parts of the work presented here.


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