Siegfried Prehn

Isolation of a protein that is essential for the first step of nuclear protein import

We have purified a cytosolic protein from Xenopus eggs that is essential for selective protein import into the cell nucleus. The purified protein, named importin, promotes signal-dependent binding of karyophilic proteins to the nuclear envelope. We have cloned, sequenced, and expressed a corresponding cDNA. Importin shows 44% sequence identity with SRP1p, a protein associated with the yeast nuclear pore complex. Complete, signal-dependent import into HeLa nuclei can be reconstituted by combining importin purified from Xenopus eggs or expressed in E. coli with Ran/TC4. Evidence for additional stimulatory factors is provided.

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope

BACKGROUND Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide. RESULTS We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. CONCLUSIONS Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation

SEC61p is essential for protein translocation across the endoplasmic reticulum membrane of S. cerevisiae. We have found a mammalian homolog that shows more than 50% sequence identity with the yeast protein. Moreover, several regions of SEC61p have significant similarities with corresponding ones of SecYp of bacteria, indicating a strong evolutionary conservation of the mechanism of protein translocation. Mammalian Sec61p, like the yeast protein, is located in the immediate vicinity of nascent polypeptides during their membrane passage. It is tightly associated with membrane-bound ribosomes, suggesting that the nascent chain passes directly from the ribosome into a protein-conducting channel. These results define Sec61p as a ubiquitous key component of the protein translocation apparatus.

Evidence for Distinct Substrate Specificities of Importin α Family Members in Nuclear Protein Import

ABSTRACT Importin α plays a pivotal role in the classical nuclear protein import pathway. Importin α shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin β-dependent import pathway. In contrast to what is found for importin β, several isoforms of importin α, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin α7. To analyze specific functions of the distinct importin α proteins, we recombinantly expressed and purified five human importin α’s along with importin α from Xenopus andSaccharomyces cerevisiae. Binding affinity studies showed that all importin α proteins from humans or Xenopus bind their import receptor (importin β) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin α proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin α3, whereas most of the other substrates were imported by all importin α proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin α proteins were revealed when two substrates were offered simultaneously.

Cloning of two novel human importin-α subunits and analysis of the expression pattern of the importin-α protein family

The import of many proteins into the nucleus is mediated by the importin‐α/β heterodimer. While only one importin‐β gene has been found, several forms of importin‐α have been described. In addition to the three human importin‐αs already identified, we report here the primary structure of two new human importin‐α proteins. The five known human importin‐α subunits can be classified into three subfamilies that appear conserved in higher eukaryotic organisms. We show by immunoblotting that the different importin‐α subfamilies are expressed in a variety of human tissues and mammalian cell lines.

Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane.

We have extended a previously developed photo‐crosslinking approach to systematically probe the protein environment of the secretory protein preprolactin, trapped during its transfer through the endoplasmic reticulum membrane. Single photoreactive groups were placed at various positions of nascent polypeptide chains of various length, corresponding to different stages of the transport process, and photo‐crosslinks to membrane proteins were analyzed. In all cases, the polypeptide segment extending from the ribosome was found to be located in a membrane environment that is formed almost exclusively from Sec61 alpha, the multi‐spanning subunit of the Sec61p complex that is essential for translocation. At early stages of the translocation process, before cleavage of the signal sequence, almost the entire nascent chain emerged from the ribosome contacts Sec61 alpha. The ‘translocating chain‐associating membrane’ protein interacts mainly with the region of the signal sequence preceding its hydrophobic core. Our results suggest that the nascent chain is transferred directly from the ribosome into a protein‐conducting channel, the major constituent of which is Sec61 alpha.

A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae.

Yeast microsomes contain a heptameric Sec complex involved in post‐translational protein transport that is composed of a heterotrimeric Sec61p complex and a tetrameric Sec62‐Sec63 complex. The trimeric Sec61p complex also exists as a separate entity that probably functions in co‐translational protein transport, like its homolog in mammals. We have now discovered in the yeast endoplasmic reticulum membrane a second, structurally related trimeric complex, named Ssh1p complex. It consists of Ssh1p1 (Sec sixty‐one homolog 1), a rather distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and accumulate precursors of secretory proteins; microsomes isolated from these cells also exhibit a reduced rate of post‐translational protein transport. Like the Sec61p complex, the Ssh1p complex interacts with membrane‐bound ribosomes, but it does not associate with the Sec62‐Sec63p complex to form a heptameric Sec complex. We therefore propose that it functions exclusively in the co‐translational pathway of protein transport.

A new phospholipid phosphatase, PRG-1, is involved in axon growth and regenerative sprouting

Outgrowth of axons in the central nervous system is governed by specific molecular cues. Molecules detected so far act as ligands that bind to specific receptors. Here, we report a new membrane-associated lipid phosphate phosphatase that we have named plasticity-related gene 1 (PRG-1), which facilitates axonal outgrowth during development and regenerative sprouting. PRG-1 is specifically expressed in neurons and is located in the membranes of outgrowing axons. There, it acts as an ecto-enzyme and attenuates phospholipid-induced axon collapse in neurons and facilitates outgrowth in the hippocampus. Thus, we propose a novel mechanism by which axons are able to control phospholipid-mediated signaling and overcome the growth-inhibiting, phospholipid-rich environment of the extracellular space.

Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1.

We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-beta, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-beta, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-alpha-dependent import of proteins with a classical NLS.

The mu2 subunit of the clathrin adaptor AP-2 binds to FDNPVY and YppØ sorting signals at distinct sites.

The endocytic sorting signal on the low‐density lipoprotein receptor for clathrin‐mediated internalization is the sequence FDNPVY in the receptors cytosolic tail. We have used a combination of surface plasmon resonance and crosslinking with a photoactivated peptide probe to demonstrate the interaction between FDNPVY‐containing peptides and the μ2 chain of purified AP‐2 clathrin adaptors (the complexes responsible for plasma membrane sorting). We show that recognition of the FDNPVY signal is mediated by a binding site in the μ2‐subunit that is distinct from the site for the more general YppØ sorting signal, another tyrosine‐based sequence also recognized by μ2‐adaptin. These results suggest the possibility that low‐density lipoprotein receptor uptake may be modulated specifically and independently of other proteins in the clathrin pathway.