Materials and Methods Yeast Subcellular Fractionation : Preparation of Enriched Nuclei and Highly Enriched NPCs

We have developed a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE proteins. Thus we found that 20% of endoplasmic reticulum (ER) marker proteins are localized at the NE. Using a novel monospecific mAb made against proteins in the H-NE fraction and found to be directed against the pore membrane protein POM152, we showed that while the majority of POM152 is localized in the NE at the NPC, a proportion of this protein is also present in the ER. This ER pool of POM152 is likely to be involved in the duplication of nuclear pores and NPCs during S-phase. Both the NEs and H-NEs were found to be competent for the in vitro posttranslational translocation of preproc~-factor. They may also be suitable to investigate other ERand NE-associated functions in cell-free systems. T HE nuclear envelope (NE) 1 defines the boundary of the nucleus in eukaryotic cells and is composed of two distinct membranes enclosing a lumenal (perinuclear) space. Facing the nucleoplasm is the inner nuclear membrane, which in higher eukaryotes is often lined by a filamentous network called the nuclear lamina. Towards the cytoplasm is the outer nuclear membrane which is continuous with the ER membranes and is thought to perform rough ER functions. The inner and the outer nuclear membranes join to form specialized circular apertures containing the nuclear pore complexes (NPCs), which regulate the exchange of material between the nucleus and cytoplasm. It is widely assumed that the NE plays a role in the control of nuclear architecture both during interphase and at mitosis. More specifically, it has been proposed that the NE could contribute in defining the spatial distribution of specific segments of the genome such as the telomeres inside the nucleus, thereby facilitating the regulation of DNA transcription and replication. Some enzymatic activities may also be restricted to the NE; for example it is likely that proteins involved in the nucleus-speAddress correspondence to Dr. G. Blobel, Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021. Ph. : (212) 327-8096; Fax: (212) 327-7880. 1. Abbreviations used in this paper. CM, crude microsome; gpaF, glycosylated pro-a-factor; H-NE, heparin-extracted NE; MT, microtubule; NE, nuclear envelope; NPC, nuclear pore complex; ppaF, prepro-a-factor; PVP, polyvinylpyrrolidone; SPB, spindle pole body. cific phosphoinositide metabolism are localized to the inner nuclear membrane. Despite considerable progress in the past few years, the molecular details of many important functions of the NE still remain poorly defined (for reviews see Hurt et al., 1992; Gilson et al., 1993; Rout and Wente, 1994; Moore and Blobel, 1994; Kilmartin, 1994). In the yeast Saccharomyces, morphometric studies indicate that the NE represents roughly 30% of the functional rough ER (Preuss et al., 1991). Thus, the isolation of NE from yeast would be useful for the study of both NE and rough ER functions. Furthermore, yeast present numerous advantages over higher eukaryotes as a system to study these functions. They have neither the complications of developmental regulation of nuclear processes, nor of nuclear disassembly, having a closed mitosis; in addition, the genetics and molecular biology of yeast are better understood than in any other eukaryote, and a large program is under way to complete the entire yeast genome sequence by the end of the decade (Maddox, 1992). Unfortunately the cell biological and biochemical characterization of cellular membranes and compartments in budding yeast remains incomplete, and would benefit from the development of rigorous cellular fractionation techniques comparable to the ones available for higher eukaryotes. We describe here a procedure for the preparation of a highly enriched NE fraction from the yeast Saccharomyces. NEs were prepared from yeast nuclei on a large scale and in high yield. To understand the relationship between peripheral and integral membrane components that define © The Rockefeller University Press, 0021-9525/95/10/19/13 $2.00 The Journal of Cell Biology, Volume 131, Number 1, October 1995 19-31 19 on M ay 4, 2017 D ow nladed fom Published October 1, 1995