The Npl4 protein is evolutionarily conserved. The sequence homology between S. cerevisiae Npl4p protein (ScNpl4p) (accession number CAA85131), S. pombe predicted protein SPBC1711.10c (SpNpl4) (accession number CAB88240), C. elegans proteins F59E12.4 (CeNpl4[1]) (accession number AAB54254) and F59E12.5 (CeNpl4[2]) (accession number AAB54255), D. melanogaster predicted protein CG4673 (DmNpl4) (accession number AAF56480), R. norvegicus (rat) Npl4 (Meyer et al., 2000), and predicted H. sapiens (human) protein FLJ20657 (HsNpl4) (accession number NP 060391) is depicted schemati-cally. The D. melanogaster Npl4 protein contains a large amino acid insertion omitted from this alignment. Ten absolutely conserved cysteine and histidine residues in the N terminus may constitute a novel Zn²⁺-finger domain. A C-terminal extension of higher eukaryotic Npl4 homologues encodes a predicted Nup153-like zinc-finger RanGDP-binding domain. The location of residues affected by mutations in the S. cerevisiae npl4-1 and npl4-2 mutants are indicated. The npl4-1 allele substitutes serine for the conserved glycine at position 323, and the npl4-2 mutation results in the production of a truncated protein lacking the C-terminal 12 amino acids.

Biochemical characterization of Npl4p localization and interactions. (A) Npl4p is found in both soluble and membrane-bound fractions. Equal protein from crude whole cell extract (lane 1), purified microsomes (lane 2), and ER/nuclear membrane-cleared extract (lane 3) derived from a wild-type yeast strain was separated by SDS-PAGE and Western blotted with anti-Npl4 antibodies. The band corresponding to Npl4p is indicated, and asterisks denote unidentified cross-reacting proteins. (B) The microsomal fraction of Npl4p is tightly membrane associated. Purified microsomes from a wild-type yeast strain were washed with either buffer alone (lanes 1 and 2), 1 M KOAc (lanes 3 and 4), 2 M KOAc (lanes 5 and 6), 3 M urea (lanes 7 and 8), 0.1 M Na₂CO₃, pH 11 (lanes 9 and 10), or 1% Triton X-100 (lanes 11 and 12). The samples were then separated into pellet (P; membrane associated; odd lanes) or soluble (S; even lanes) fractions and subjected to SDS-PAGE and Western blotting with anti-Npl4 and anti-Cdc48 antibodies. The extraction profiles of the peripheral ER membrane protein Sec17p and the ER integral membrane protein Sec62p were determined by Western blotting with anti-Sec17 and anti-Sec62 antibodies as controls. (C) Npl4p-protein A (pA) copurifies with Cdc48p and Ufd1p. Yeast whole cell extracts derived from either a strain expressing untagged Npl4p (lane 1) or Npl4p-pA (lane 2) were incubated with IgG-Sepharose beads. After extensive washing, proteins bound to the beads were eluted with acid and visualized by SDS-PAGE and Coomassie blue staining. Mass spectral analysis of excised protein bands revealed the identity of the Npl4p-pA copurifying p100 and p43 proteins as Cdc48p and Ufd1p, respectively.

NPL4 function is required for OLE1 transcription. Northern analysis was performed on total RNA isolated from ole1Δ (lane 1), wild-type (WT; lanes 2–4), npl4-1 (lanes 5–7), npl4-2 (lanes 8–10), cdc48-3 (lanes 11–13), ufd1-1 (lanes 14-16), and rpn4Δ cells (lanes 17–19) with OLE1- and ACT1-specific DNA probes. Cells were grown in YEPD media at 25°C with or without supplemented UFAs. For wild-type cells, the temperature-sensitive-strains npl4-1, npl4-2, and cdc48-3, and non–temperature sensitive strains ufd1-1 and rpn4Δ, samples were also collected after shifts to 37°C for 60 min. A normalized OLE1/ACT1 ratio was calculated (see MATERIALS AND METHODS) for each sample to allow for quantitation of the relative changes in OLE1 expression among samples.

A model for Npl4 function in the proteasome-dependent processing of the ER membrane-bound transcription factors Mga2p and Spt23p. Details are presented in the text.

Npl4p-GFP localizes to ER/nuclear membranes. (A) Anti-Npl4 (left) and anti-GFP (right) Western blots of whole cell extracts derived from strains expressing endogenous Npl4p (lanes 1 and 4), a genomically tagged Npl4p-GFP (lanes 2 and 5), and a genomically tagged Npl4p-protein A(pA) (lanes 3 and 6). The anti-Npl4 blot shows the precise replacement of endogenous Npl4p with the two different C-terminally tagged proteins (compare lanes 2 and 3 to lane 1). The robust reactivity of anti-Npl4 with Npl4-pA (lane 3) is a result of antibody interactions with Npl4p epitopes as well as the IgG-binding pA tag. The rabbit anti-GFP blot recognizes both Npl4p-GFP and Npl4p-pA (lanes 5 and 6, respectively). The identity of the different proteins is indicated by arrows. The asterisk indicates an unidentified cross-reacting protein. (B) Npl4p-GFP localization as observed by fluorescence microscopy. The live cells were viewed at midlog phase. Localization is mostly nuclear (white arrowhead) and cytoplasmic, with an apparent concentration at perinuclear membranes. The white arrow indicates an example of Npl4p-GFP localization to peripheral membranes. A Nomarski image of the cells is shown to the right. (C) Npl4p-GFP colocalization with DAPI-stained nuclear DNA. Cells were fixed in formaldehyde, permeabilized with Triton X-100, and DAPI stained before fluorescence microscopy. The majority of Npl4p-GFP localizes to a region overlapping with and surrounding the nucleus as observed by DAPI staining. A Nomarski image of the cells is shown to the right. (D) Npl4p-GFP expression as viewed by fluorescence microscopy on a DeltaVision platform. Sequential 0.1-μm fluorescence images were taken through a diploid yeast strain expressing Npl4p-GFP from both NPL4 loci and subjected to five rounds of deconvolution. The images presented represent a central plane from deconvolved data. The white arrowhead indicates an example of Npl4p-GFP concentration at perinuclear membranes. White arrows indicate examples of Npl4p-GFP localization to peripheral membranes.

Yeast npl4 mutants are rescued by regulators of the UFA pathway and the proteasome. (A) Suppression of npl4-1 and npl4-2 temperature sensitivity by high-copy expression of OLE1, SPT23, MGA2, and RPN4. npl4-1 (PSY2340, top) and npl4-2 (PSY2341, bottom) strains transformed with either empty vector, or 2 μ vectors containing NPL4, OLE1, MGA2(aa1-914), MGA2, SPT23, or RPN4 were serially diluted onto YEPD media. The number of cells spotted in each dilution is indicated at the bottom. Plates were incubated at the indicated temperatures for 24–48 h. (B) Temperature sensitivity of a npl4-1 strain is extragenically suppressed by a transposon insertion allele of SPT23. Growth of wild-type (WT; FY23), npl4-1 (PSY2373), and npl4-1 SPT23:: Tn3:: LEU2 (PSY2377) strains on Leu⁻ plates at the indicated temperatures. The FY23 and PSY2373 strains were transformed with an empty LEU2 vector to allow growth on Leu⁻ dropout plates. (C) Schematic of the S. cerevisiae Mga2p and Spt23p proteins. These partially redundant factors are 38% identical and 54% similar. Sequence features include a putative basic nuclear localization signal (NLS), a putative leucine-rich nuclear export signal (NES), and two ankyrin repeats. The TIG domain is an Ig-like fold domain found in cell surface receptors and intracellular transcription factors including NF-κB. The C-terminal transmembrane domain (TM) is indicated. Blocks of strong amino acid conservation without assigned or predicted function are indicated by white boxes. The site of truncation in the isolated MGA2 high-copy suppressor clone (pPS2019) is indicated by an arrow in the Mga2p schematic. The site of transposon (Tn::LEU2) insertion in the npl4-1 extragenic suppressor allele of SPT23 (PSY2377) is indicated in the Spt23p schematic. (D) Temperature sensitivity of yeast npl4 mutants is partially rescued by supplementing growth media with UFAs. Wild-type (WT), ole1Δ, npl4-1, and npl4-2 strains were struck onto YEPD plates unsupplemented (left) or supplemented (right) with 0.5 mM UFAs (0.25 mM palmitoleic acid [16:1], 0.25 mM oleic acid [18:1], 1% Tergitol). Plates were incubated at the indicated temperatures for 48 h.

NPL4 is required for efficient processing of ubiquitinated Mga2p and Spt23p fusion proteins. (A) Induction of GFP-Mga2p fusion protein in wild-type (WT; lanes 1–4), npl4-1 (lanes 5–7), npl4-2 (lanes 8–10), cdc48-2 (lanes 11–13), ufd1-1 (lanes 14-16), and rpn4Δ (lanes 17–19) cells at 25°C. Cells were collected for analysis by anti-GFP Western blot at the indicated time points into galactose induction. Asterisks indicate higher molecular weight GFP-Mga2p proteins. (B) Induction of GFP-Spt23p fusion protein in wild-type (WT; lanes 1–4), npl4-1 (lanes 5–7), npl4-2 (lanes 8–10), cdc48-2 (lanes 11–13), ufd1-1 (lanes 14-16), and rpn4Δ (lanes 17–19) cells at 25°C. Cells were collected for analysis by anti-GFP Western blot at the indicated time points into galactose induction. Asterisks indicate higher molecular weight GFP-Spt23p proteins. (C) Subcellular localization of GFP-Spt23p in wild-type (WT), npl4-1, npl4-2, cdc48-2, ufd1-1, and rpn4Δ cells after 120-min galactose inductions at 25°C. GFP-Spt23p signal is largely nucleoplasmic in wild-type cells, while remaining largely ER/nuclear envelope associated in the mutant strains. (D) GFP-Spt23p (left) and GFP-Mga2p (right) were immunoprecipitated (IP) from wild-type (WT) or npl4-1 yeast whole cell extracts expressing Myc-tagged ubiquitin. Expression of GFP-Spt23p and GFP-Mga2p was either induced for 2 h with galactose (lanes 2, 4, 6, 8, 10, 12, 14, and 16), or repressed with the addition of glucose (lanes 1, 3, 5, 7, 9, 11, 13, and 15) before immunoprecipitation. Samples run on the same gel were analyzed by anti-GFP (lanes 1–4 and 9–12) or anti-Myc (lanes 5–8 and 13–16) Western blotting.