Mutations in the clathrin-assembly Picalm are responsible for the hematopoietic and iron metabolism abnormalities in fit1 mice

Mitchell L. Klebig*†‡, Melissa D. Wall*†, Mark D. Potter§¶, Erica L. Rowe§, Donald A. Carpenter†, and Eugene M. Rinchik*†

*Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996; †Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6445; and §University of Tennessee–Oak Ridge National Laboratory, Graduate School of Genome Science and Technology, Oak Ridge, TN 37830-8026

Communicated by Liane B. Russell, Oak Ridge National Laboratory, Oak Ridge, TN, May 2, 2003 (received for review February 6, 2003) Recessive N-ethyl-N-nitrosourea (ENU)-induced mutations recov- involved in adaptor protein complex 2 (AP2)-dependent ered at the fitness-1 (fit1) in mouse 7 cause clathrin-mediated endocytosis in the cell (12–14). The mutations hematopoietic abnormalities, growth retardation, and shortened disrupt different splice-donor sites and result in transcripts that life span, with varying severity of the defects in different . are missing exons and are predicted to truncate Picalm . Abnormal iron distribution and metabolism and frequent scoliosis This finding demonstrates that Picalm and likely the clathrin- have also been associated with an of intermediate severity mediated endocytic processes involving it play important roles in (fit14R). We report that fit14R, as well as the most severe fit15R hematopoiesis, iron metabolism, and growth. allele, are nonsense point mutations in the mouse ortholog of the human phosphatidylinositol-binding clathrin assembly protein Materials and Methods (PICALM) gene, whose product is involved in clathrin-mediated Mice. The origin and initial maintenance of mouse strains car- endocytosis. A variety of leukemias and lymphomas have been rying the fit14R and fit15R mutations, as well as the generation of associated with translocations that fuse human PICALM with the mice hemizygous for the fit1 alleles [i.e., Tyrc fit1͞Del(Tyr fit1–5R putative transcription factor gene AF10. The Picalm and fit1)26DVT, hereafter referred to as fit1͞Del26DVT], have been fit1–4R Picalm mutations are splice-donor alterations resulting in described previously (2, 8, 15, 16). Tyrc-ch ϩ͞Del26DVT mice are transcripts that are less abundant than normal and missing exons phenotypically normal (2). The fit1 alleles analyzed in this study 4 and 17, respectively. These exon deletions introduce premature were induced on a BALB͞cRl chromosome and are maintained termination codons predicted to truncate the proteins near the N as Tyrc fit1 ϩ͞Tyrc-ch ϩ fr heterozygotes by mating them each and C termini, respectively. No mutations in the encoding generation to inbred FRCH͞R(Tyrc-ch ϩ fr͞Tyrc-ch ϩ fr) mice. Picalm, clathrin, or components of the adaptor protein complex 2 (AP2) have been previously described in which the suite of disor- Bacterial Artificial Chromosome (BAC) Isolation and Analysis. BAC fit1 ders present in the Picalm mutant mice is apparent. These clone RPCI-23-68G17, derived from a C57BL͞6J library (17), mutants thus provide unique models for exploring how the endo- was purchased from ResGen͞Invitrogen; BAC DNA was iso- cytic function of mouse Picalm and the transport processes medi- lated for analysis according to the manufacturer’s instructions. ated by clathrin and the AP2 complex contribute to normal hema- SSLP markers D7Mit32, D7Mit352, and D7Mit300; STS markers topoiesis, iron metabolism, and growth. M-09035 and M-02716; and markers for the D7Cwr11D, D7Mlk1 and D7Mlk2 loci, all of which map within the deletions and yeast ͞ haracterization of mutations causing anemia and or iron artificial chromosome (YAC) contigs spanning the fit1 interval ͞ Cmetabolism transport defects has proven to be a valuable (15, 18, 19), were mapped against the BAC by PCR analyses, by approach for studying the development and regulation of both using specific primers. SSLP primers were purchased from the hematopoietic system and body-iron content (e.g., refs. 1–7). ResGen͞Invitrogen, and the sequences of all other primers used Mice hemi-or homozygous for N-ethyl-N-nitrosourea (ENU)- in this study are available (Table 1, which is published as induced mutations at the fit1 locus have shortened life span, supporting information on the PNAS web site, www.pnas.org). severe runting, microcytic hypochromic anemia, lowered white BAC DNA was physically sheared and subcloned into plasmids, blood cell counts, and reduced erythroid and myeloid progenitor which were then sequenced (4,028 sequence reads). cell populations during both fetal and adult hematopoiesis (2, 8). 5R Additional study of the most severe allele, fit1 (formerly RT-PCR Analysis of Picalm cDNA. 4397SB Total RNA was isolated from fit1 ), revealed reticulocytosis, extramedullary erythropoi- various tissues of mice as described (20). First-strand cDNA was esis in the spleen, iron deposition in the liver, and B cell synthesized from Ϸ3 ␮g of total RNA, by using both oligo-dT deficiency (2). Detailed study of an allele of intermediate 4R 4226SB and random decamer priming, with the RETROscript Kit severity, fit1 (formerly fit1 ), showed that the anemia was (Ambion, Austin, TX), following the manufacturer’s instruc- mildly regenerative and demonstrated functional iron deficiency, abnormal iron distribution suggestive of impaired iron transport from the liver to other tissues, biochemical evidence of liver Abbreviations: PICALM, phosphatidylinositol-binding clathrin assembly protein; dysfunction, increased myeloid͞erythroid ratios in the bone CHC, clathrin heavy chain; AP2, adaptor protein complex 2; BAC, bacterial artificial marrow, and scoliosis and lumbar vertebral abnormalities (9– chromosome; YAC, yeast artificial chromosome. 11). The fit1 mutants exhibit alterations in hematopoiesis, iron Data deposition: The BALB͞cRl Picalm cDNA sequence was deposited in the GenBank database (accession no. AY206701); the entire sequence shown in Fig. 3 is available in the metabolism, and bone growth, unlike other animal models of Third Party Annotation Section of the DNA Data Bank of Japan (DDBJ)͞EMBL͞GenBank inherited microcytic hypochromic anemia (11). Moreover, they databases (accession no. TPA: BK001028). may also be a unique model for the study of scoliosis (11). ‡To whom correspondence should be addressed at: Oak Ridge National Laboratory, P.O. We report here that both of these fit1 alleles are point Box 2008, Bethel Valley Road, Oak Ridge, TN 37831-6445. E-mail: [email protected]. mutations in the mouse phosphatidylinositol-binding clathrin ¶Present address: Molecular Genetics Department, Lexicon Genetics, Inc., The Woodlands, assembly protein (Picalm) gene, which encodes an assembly TX 77381-1160.

8360–8365 ͉ PNAS ͉ July 8, 2003 ͉ vol. 100 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1432634100 Downloaded by guest on September 28, 2021 tions. The resulting cDNAs were amplified by PCR with the use of specific primers (see Fig. 3A and Table 1) and, to reduce the incorporation of Taq-induced errors, a 9:1 mixture of Taq (AmpliTaq, Perkin–Elmer) and PfuTurbo (Stratagene) poly- merases. The final PCR reaction contained RT cDNA (Ϸ750 ng), specific primers (0.2 ␮M each), dNTPs (200–250 ␮M each), ␮ and MgCl2 (1.5–1.75 mM) in a total volume of 25–50 l. In general, 30–35 PCR cycles of denaturation at 94°C for 30 s, annealing at 52–59°C for 30 s, and extension at 72°C for 30–40 s were performed. Some amplifications were performed by including 10 touchdown-PCR cycles before the general cycling conditions. RT-PCR products either were sequenced directly with the same primers used for the PCR after ExoSAP-IT (United States Biochemical) treatment or were cloned (TOPO-TA Cloning Kit, Invitrogen Life Technologies) and then sequenced with vector primers. DNA sequencing was performed in both forward and Fig. 1. Genetic and physical map of a segment of mouse chromosome 7 reverse directions with the Big Dye Terminator Cycle Sequenc- containing the fit1 locus. Chromosome 7 is indicated by the line with the oval ing Kit (Applied Biosystems), according to the manufacturer’s (depicting the centromere) at its left end. Relative locations of genetic loci instructions. After ethanol precipitation, the reaction products (italicized) and STS markers (M-09035 and M-02716) are shown above the were analyzed with an ABI 377 sequencing system (Applied chromosome. No physical distance is implied by placement of any of the Biosystems). markers. Bold lines below the chromosome represent Del(Tyr) deletions, and their allele names (21) are shown (Left). The minimal deletion interval con- Mutation Analysis. RT-PCR primers were designed, on the basis taining the fit1 locus (15) is indicated. A YAC contig spanning part of the of cDNA sequence assembled from all of the ESTs, to amplify region is shown above the chromosome [390-kb YAC clone D6E7 (18); WC7.32 ͞ ͞ overlapping Ϸ500-bp fragments that together spanned the entire contig is described at www.informatics.jax.org searches contig.cgi?1251 (660 kb from D7Mit32 to M-02716)]. D7Rn5 and D7Cwr11D are loci derived Picalm ORF and were predicted, on the basis of the EST from the indicated deletion breakpoints. The RPCI-23-68G17 BAC clone (194 assembly, not to be alternatively spliced. Normal and size-altered kb) containing the Picalm gene (with the transcriptional orientation shown) RT-PCR products obtained from liver RNAs derived from and several markers is shown above the chromosome. The lines projecting wild-type and mutant mice (three individuals of each) were from the ends of the BAC to the chromosome indicate that the BAC is located cloned and sequenced. Nucleotide substitutions in genomic between, but does not include, the D7Rn5 and D7Mit352 loci. The portion of DNA responsible for cDNA alterations were identified by the BAC between its distal end and the dashed͞dotted line extending from the amplifying fragments containing the affected exons and splice D7Mit32 locus indicates the minimum part of the BAC known to be located junction sequences from 30–60 ng of genomic DNA template, within the fit1 interval.

using the same general conditions as described above for RT- GENETICS PCR, and sequencing the PCR products. To characterize the (Applied Biosystems), respectively. Identities and similarities fit15R mutation, primers 181 and 182, corresponding to se- quences in introns 3 and 4, were used to amplify a 388-bp product between the mouse and human cDNA and amino acid sequences containing exon 4 and its adjacent splice-site sequences from were determined, and sequence alignments were performed, by fit15R͞Del26DVT DNA. For the fit14R allele, primers 214 and 215, analyses with BLAST and MACVECTOR (CLUSTALW alignment) designed from introns 16 and 17, were used to amplify a 415-bp software. product containing exon 17 and its flanking splice sites from Results fit14R͞Del26DVT DNA. Identification of the Mouse Picalm Gene at the fit1 Locus. We Probe Hybridizations. A mixture of the 147͞148 and 149͞150 previously (15) used a series of Tyr deletions to define a RT-PCR products (probe A) was column purified, labeled with minimum deletion interval, between the eed and D7Cwr11D loci, ALL-IN-ONE Random Prime DNA Labeling Mix (Sigma), and that contains fit1, and we identified a YAC contig containing this hybridized to the Northern blot. DNA segment (Fig. 1; ref. 18). In the search for fit1 candidates, two unique-sequence DNA clones that mapped within the Bioinformatic Analyses. BLAST (genome survey sequence data- minimal deletion interval, Y6CD3 and C58 (from the D7Mlk1 base) analysis (www.ncbi.nlm.nih.gov͞BLAST) of the Y6CD3 and D7Mlk2 loci, respectively; data not shown), were isolated sequence identified end-sequence from BAC RPCI-23–68G17. from YAC clone D6E7 and sequenced. Database searching The sequence trace data from the BAC were analyzed, assem- indicated that part of the Y6CD3 sequence was complementary bled, and edited with the PHRED, PHRAP, and CONSED programs to end sequence from the BAC clone RPCI-23-68G17. PCR (www.phrap.org). Murine ESTs with homology to human and rat typing determined that the BAC also contains the D7Mlk2 and PICALM cDNAs (GenBank accession nos. NM࿝007166, D7Mit32 markers (data not shown), both of which map within the XM࿝165625, AF041374, and AF041373) were also identified by deletion interval containing fit1 (Fig. 1). The BAC was se- BLAST analysis. ARTEMIS COMPARISON TOOL (www.sanger.ac.uk͞ quenced, and BLAST analysis of the sequence revealed the software͞ACT), CONSED, and BLAST were used to determine the presence of the mouse ortholog of the human PICALM (for- correct order and orientation of the BAC sequence contigs and merly CALM) gene (12). PICALM maps to HSA chromosome the exon–intron boundaries of Picalm by alignment of the BAC 11q14 and appears to be a ubiquitously expressed paralog of the sequences with the mouse consensus cDNA sequence made SNAP91 gene, which encodes the synaptic clathrin-assembly from EST assemblies. Sequence editing of PCR products (and protein AP180 (12, 22). A number of leukemias (both acute clones of PCR products) and the assembly of the PCR sequences lymphocytic and acute myeloid) and lymphomas have been and ESTs (see Table 2, which is published as supporting infor- reported in which the human PICALM gene forms fusion mation on the PNAS web site, for GenBank accession numbers transcripts with the putative transcription factor AF10 in HSA and sources of these ESTs) into contiguous cDNA sequences, chromosome 10 by virtue of t(10, 11) translocations (e.g., refs. were performed with FACTURA and AUTOASSEMBLER software 23–25). AP180 and PICALM function in the recruitment of

Klebig et al. PNAS ͉ July 8, 2003 ͉ vol. 100 ͉ no. 14 ͉ 8361 Downloaded by guest on September 28, 2021 clathrin and AP2 to cell membranes at sites of coated-pit formation and clathrin-vesicle assembly (13, 14, 26, 27). The location of the mouse Picalm gene, and the fact that clathrin͞AP2-mediated endocytosis of the transferrin͞ transferrin–receptor complex from the plasma membrane is the main mechanism of iron uptake into most cells (28, 29), sug- gested that Picalm was a good candidate for fit1, the only locus associated with severe anemia that was identified in this mu- tagenized chromosomal region (2, 8). Thus, we first assembled a composite mouse Picalm cDNA sequence of Ϸ3.6 kb (Fig. 2A and data not shown) from numerous murine ESTs and partial cDNAs that were identified by homology to human and rat PICALM cDNAs (12, 30). BLAST analysis of the murine ESTs in GenBank also indicated the potential for several alternatively spliced Picalm cDNA isoforms (Fig. 2A).

RT-PCR Analysis and Sequencing of the Wild-Type Mouse Picalm cDNA. PCR primers based on the consensus composite cDNA sequence predicted from the murine ESTs were used to amplify six overlapping segments of the Picalm cDNA (Fig. 2B) from liver and spleen RNAs derived from BALB͞cRl mice, which carry the wild-type fit1 allele. RT-PCR products of expected sizes were obtained with all primer pairs, and one or more additional products were obtained with the 151͞152 and 153͞154 primer pairs (Fig. 2B), in agreement with the expectation of several different alternatively spliced isoforms of the Picalm mRNA. All of the RT-PCR products were cloned and sequenced, and the sequences were assembled into versions of Picalm cDNA that encode variant forms of the Picalm protein (Figs. 2A and 3A). The largest potential cDNA assembled from these products, which includes all of the alternatively spliced segments, is a 2.4-kb cDNA sequence containing a complete ORF predicted to encode a 660-aa protein (Fig. 3A). Comparison of the compre- hensive Ϸ3.6-kb cDNA and BAC sequences revealed that the mouse Picalm gene is comprised of 21 exons (Figs. 2A and 3A; Fig. 2. Mouse Picalm mRNA map and effects of fit1 mutations on the gene. (A) exon sizes and exon–intron junction sequences are shown in Map of a composite 3,660-bp mouse Picalm mRNA predicted from the murine Table 3, which is published as supporting information on the ESTs and partial cDNAs homologous to the human and rat PICALM cDNAs. The PNAS web site). portion of the mRNA encoding the protein (ORF) is indicated by the thick black The sequence analyses also showed that the multiple RT-PCR arrow above the map. The locations of the primers used in RT-PCR analyses are products obtained with the 151͞152 and 153͞154 primer pairs indicated above the map along with the numeric primer labels. The segment of (Fig. 2B) represented alternatively spliced forms of the transcript the cDNA shown by the stippled rectangle above the map (Probe A) was used as in which exons 13 and 18 and the first 15 bp of exon 15 are present a probe on the Northern blot (D). The boundaries of the 21 Picalm exons in the in various combinations (Figs. 2A and 3). The omission of any cDNA are shown below the map. Alternatively spliced exons or exon segments in of these alternatively incorporated cDNA segments in the Picalm the wild-type gene are designated by the gray rectangles, and the exons that are missing in mRNAs derived from the fit15R and fit14R alleles are indicated by the transcript (in all possible combinations) still preserves the read- black boxes below the map. (B) Picalm RT-PCR products separated by agarose gel ing frame, so that all of the possible alternative transcripts can electrophoresis. The RNA samples analyzed are indicated above each lane: B, encode the remainder of the C-terminal residues of the protein BALB͞cRl (ϩ͞ϩ) control; 5R, fit15R͞Del26DVT; 4R, fit14R͞Del26DVT. RT-PCR primer (Fig. 3A). Due to the different possible transcript forms, the pairs are shown above the lane headings. The sizes of the molecular weight normal protein could range from 597 to 660 aa in length. No marker (M) bands are shown at left for the 396͞146 RT-PCR products and in the ESTs or cDNAs that would encode the largest potential protein middle for the other products. Abnormal-sized RT-PCR products obtained from were identified in the sequence databases, whereas several that mutant RNAs (asterisks): fit15R, 394-bp 147͞148 product; fit14R, 407-, 398-, and would encode a Յ655-residue protein can be found. Therefore, 383-bp 153͞154 products. The sequence of the 460-bp 151͞152 RT-PCR product it is not yet known whether the complete 660-residue protein (the middle band of the three bands obtained) revealed it is derived from another gene (Fpgs) that is unrelated to Picalm except for part of the primer sequences. shown in Fig. 3 is actually produced in vivo. If it is, it is likely a (C) Summary of the alterations caused by the two N-ethyl-N-nitrosourea (ENU)- minor product of the gene. induced mutations at the genomic DNA and mRNA levels. Exons are indicated by BLAST analysis of this mouse cDNA sequence identified two numbered boxes. The relevant wild-type splice acceptor (AG) and splice donor human cDNA (GenBank accession nos. NM࿝007166 and (GT) sites (Left Upper and Lower) and the mutated splice donor sequences (Right) XM࿝165625) and many EST sequences, which were then assem- are shown. The splicing events leading to the mRNA products shown are indi- bled into a comprehensive human cDNA sequence that includes cated by the dashed lines. In each case, BALB͞cRl represents the wild-type splicing all alternatively spliced segments, including the same three pattern. (D) Hybridized Northern blot (Upper) showing the major Ϸ3.6-kb Picalm regions identified in mouse and five additional regions that are transcript in control and mutant mice. The blot contains total RNAs from the skins apparently unique to human (sequence alignments shown in Fig. and livers of 1-week-old mice with genotypes indicated above each lane, as defined in B. The blot was hybridized with Probe A (A). A loading control 4, which is published as supporting information on the PNAS (ethidium-bromide-stained RNA after transfer to the membrane) is shown below web site). BLAST comparison of the largest potential mouse (Fig. the autoradiogram (18S and 28S ribosomal RNA bands are indicated). If the level 3A) and human PICALM cDNA sequences revealed an identity of expression from each mutant fit1 allele was the same as that from the of 94.5% in the ORF and 91% overall (Fig. 4). The amino acid wild-type (BALB͞cR1) allele, then the signal intensity of the transcript detected in sequences of the mouse and human proteins predicted from the 5R and 4R lanes (RNAs from hemizygous mutants) is expected to be only 50% these cDNAs are 98.5% similar and 97.7% identical, and both of the signal intensity of the wild-type transcript in the B lane.

8362 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1432634100 Klebig et al. Downloaded by guest on September 28, 2021 include all of the defined functional domains͞motifs (amino acid sequence alignments shown in Fig. 5, which is published as supporting information on the PNAS web site), indicating Picalm is highly conserved. Hybridization of a Picalm cDNA probe (Fig. 2A) to a North- ern blot containing BALB͞cRl RNAs identified a predominant transcript of Ϸ3.6 kb in several tissues (Fig. 2D and data not shown), consistent with the ubiquitously expressed human gene (12). The mouse cDNA predicted from the available ESTs is also Ϸ3.6 kb in length (Figs. 2A and 3A), suggesting the composite sequence represents a full-length transcript.

Two fit1 Mutations Lead to Picalm Transcripts That Are Missing Exons and Predicted to Encode Truncated Proteins. The primers used to amplify the six overlapping segments of the wild-type Picalm cDNA were also used to amplify these segments from liver RNAs derived from fit1͞Del26DVT hemizygotes. Most of the RT-PCR products obtained from the mutants were the same size as from wild type; however, smaller-than-normal products, but no wild- type-sized products, were obtained from mutant fit15R͞Del26DVT and fit14R͞Del26DVT mice with the 147͞148 and 153͞154 primer pairs, respectively (Fig. 2B). Cloning and sequencing of these mutant-specific RT-PCR products revealed missing exons in the mutants (exon 4 in fit15R and exon 17 in fit14R) (Figs. 2 A and C and 3A), and that both alternatively spliced regions within the 153͞154 amplicon are correctly incorporated into the size- altered fit14R-derived transcripts. The relative abundance of the different alternatively spliced 153͞154 RT-PCR products ob- tained from the fit14R mutants also appears to be similar to that in wild-type (Fig. 2B). In each mutant, the absence of the exon in the mRNA leads to a frameshift in the ORF 3Ј of the missing exon and, as a result, the incorporation of a few aberrant codons followed by a premature termination codon. Thus, the mRNAs GENETICS

(Table 2) derived from these regions of the cDNA. The sequences of the exons missing in the transcripts produced by the fit15R and fit14R alleles (exons 4 and 17, respectively) are highlighted by dark gray shading, and the last normal amino acid residue of the putative truncated protein encoded by each allele is circled (unshaded and shaded for fit15R and fit14R, respectively). The se- quences of the alternatively spliced exons (exons 13 and 18, and the first 15 bp of exon 15) defined in this study are shown underlined and in bold. The polyadenylation signal (AATAAA) is underlined twice. The amino acid residues of the epsin N-terminal homology domain (residues 20–145) are shown in bold, and the amino acids implicated in the binding of membrane PtdIns(4,5)P2 (14) are enclosed by open boxes. Enclosed by light-gray boxes are the amino acid sequence DPF, a motif that is known to interact with the ␣-adaptin appendage domain of AP2 (27, 31, 32), and two NPF motifs, which interact with protein-binding modules (Eps15 homology domains) found in several proteins that are also part of the endocytic machinery (31, 33). Putative type I and II clathrin-binding sequences (CBS-I and -II) (34) are indicated by the diagonally striped boxes. (B) Full-length and truncated Picalm proteins pre- dicted to be encoded by the wild-type (ϩ) and two mutant transcripts (below, fit1 mutant alleles shown to the left). The largest predicted form of the wild-type protein (660 residues) is shown here and would be produced by incorporation of all three alternatively spliced segments [indicated by the Fig. 3. Picalm cDNA and proteins. (A) Sequences of the mouse Picalm cDNA black boxes shown below the schematic of the protein and encoded by exon and protein [sequence is available in the Third Party Annotation Section of the 13 (E13), exon 18 (E18), and the first 15 bp of exon 15 (E15)]. The number at the DNA Data Bank of Japan (DDBJ)͞EMBL͞GenBank databases under the acces- end of each truncated protein indicates the amino acid residue at which the sion no. TPA: BK001028]. The predicted amino acid sequence of the encoded normal sequence ends; the amino acid sequences shown to the right of the protein is shown below the cDNA sequence. The cDNA sequence is numbered truncated protein are extra frameshift-induced residues added to the C- according to the largest potential cDNA that was assembled from the mouse terminal end of the protein before a stop codon (asterisk) occurs. Gray-shaded ESTs͞cDNAs homologous to the human and rat cDNAs and includes all of the area, epsin N-terminal homology (ENTH) domain; stippled areas, regions of alternatively spliced segments. The exon junctions are indicated by ; the highest homology with AP180 proteins (13). The locations of the DPF and NPF locations of PCR primers in the sequences are shown by arrows above the cor- motifs, and the putative type I and II clathrin-binding sequences (CBS-I and -II) responding sequence. The 2,398 bp of sequence shown between primers 396 in the normal protein are shown. Three different segments of the protein and 156 was derived from the inbred control BALB͞cRl strain used in this study (labeled as A, B, and C) are known to contain distinct sites of interaction with (GenBank accession no. AY206701) and represents the assembly of the CHC, the major binding partner of PICALM (13). The C-terminal portion from sequences of the different RT-PCR products obtained from BALB͞cRl with all residues 414–660 (segments B and C) has a much stronger interaction with six primer pairs (Fig. 2B). The sequences located 5Ј and 3Ј of this segment CHC than the N-terminal segment A (residues 1–413); the last 40 residues are the consensus sequences obtained from an assembly of the mouse ESTs (segment C) are essential for binding CHC with high affinity (13).

Klebig et al. PNAS ͉ July 8, 2003 ͉ vol. 100 ͉ no. 14 ͉ 8363 Downloaded by guest on September 28, 2021 obtained from the mutant fit15R and fit14R alleles are predicted unc-11 are known to interfere with clathrin-dependent recycling to encode proteins truncated at residues 116 and 559, respec- of synaptic vesicles and the regulation of vesicle size and number tively (Fig. 3B). Northern blot analysis also revealed that mice (37, 38). Because of its strong affinity for clathrin, colocalization hemizygous for these fit1 mutations express a Picalm transcript with a major depot of clathrin-coated vesicles, and almost close in size to that in wild-type mice but at a level considerably complete colocalization in the cell with AP2, but not AP1, lower than the wild-type level, which was particularly evident in PICALM has been implicated in the recruitment of clathrin and the fit14R͞Del26DVT mutants (Fig. 2D). Most likely, this is due to AP2 to cellular membranes for the formation of nonsynaptic nonsense mutation-mediated mRNA decay (35) and will also vesicles (13). Moreover, because AP180 is thought to regulate lead to reduced levels of the predicted truncated proteins. synaptic vesicle size and number (37, 38), its close paralog, To confirm the two fit1 mutations at the genomic level, the PICALM, may be involved in the similar regulation (27) of affected exons (exons 4 and 17) and their flanking intronic nonsynaptic vesicles. Decreased serum levels and abnor- sequences containing the splice acceptor and donor sites were mal tissue distribution of iron in mice hemizygous for the amplified by PCR from genomic DNA of the mutant and Picalmfit1–4R mutation suggest that iron transport from the liver wild-type animals. Sequencing of these PCR products revealed to other tissues is impaired (10). Because erythroid hemoglobin that, in both mutant alleles, the splice donor site immediately synthesis places particularly heavy demands on the transferrin following the affected exon was altered from a GT to a GA, cycle (39), decreased efficiency of AP2-dependent clathrin- which leads to the exon deletion observed in the mRNA by mediated endocytosis due to a mutational defect in the predicted splicing via the splice donor for the preceding exon (Fig. 2C). recruitment and vesicular-regulation functions of Picalm in the mutant mice could have a larger impact on transferrin endocy- Discussion tosis and subsequent hemoglobinization than on nonhematopoi- We have observed that two mouse fit1 mutations of differing etic functions, a hypothesis that can now be tested rigorously. phenotypic severity disrupt independent splice-donor sites and The most phenotypically severe mutant allele, Picalmfit1–5R, result in Picalm transcripts with missing exons and of lower has the potential to encode a protein that is truncated after abundance than normal. Due to the incorporation of premature amino acid residue 116 (removing 82% of the amino acid termination codons downstream of the missing exons, the tran- sequence). This truncated protein would lack all of the defined scripts produced from the severe and milder alleles are predicted structural motifs known to interact with clathrin, the AP2 to specify proteins truncated near the N and C termini, respec- complex, and other endocytic proteins (13, 27, 31–34). It would tively. This finding indicates that Picalm is the fit1 gene (now also be missing the C-terminal Ϸ20% of the epsin N-terminal designated Picalmfit1). Because no other in vivo disorders have homology domain (Fig. 3), which mediates associations with cell been associated with recessive mutations in Picalm, the Picalmfit1 membranes and clathrin vesicle-mediated internalization of re- mutants present unique models for exploring the role of the ceptor–ligand complexes via interactions with membrane phos- mouse Picalm protein in the clathrin-mediated endocytic pro- phatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] (14, 40). The cesses that are essential for hematopoiesis, iron uptake͞ major binding partner of human PICALM is clathrin heavy chain metabolism, and growth. (CHC), although weaker interactions among PICALM and the The human PICALM protein and its neuron-specific paralog, ␣-adaptin subunit of AP2 and several other uncharacterized AP180, are a part of the endocytic machinery involved in the proteins have been reported (13). Three different segments of AP2-directed formation of clathrin-coated vesicles from cell PICALM are known to contain sites of interaction with CHC membranes, but appear to participate in the formation of distinct (Fig. 3B), but the last 40 C-terminal residues have the highest types of coated vesicles (13, 14, 26, 27). Disruption of individual affinity (13) despite the absence of any currently defined clath- AP2-subunit genes arrests development during embryonic stages rin-binding sequences (34) in that segment (Fig. 3). Thus, in both Drosophila melanogaster and Caenorhabditis elegans (36). similarly to AP180 (27), PICALM must interact with CHC via A null mutation of the D. melanogaster SNAP91 ortholog (lap) additional as yet undefined structural motif(s) in this C-terminal is lethal at the early larval stage, although some escapers survive part of the protein. We have also found that the transcript to the pupal and even adult stages (37). Null mutations of the C. produced from the milder Picalmfit1–4R allele has the potential to elegans SNAP91 ortholog, unc-11, are viable with various loco- encode a truncated protein that is missing only the C-terminal motion defects (38). We have now shown that the ubiquitously 101-residue portion containing the highest-affinity CHC binding expressed paralog of SNAP91 in mammals, Picalm, is not essen- site(s) and one of the NPF motifs. tial for early embryogenesis but is necessary for juvenile survival. Generation of an antibody to the very N terminus of mouse The more specific hematopoietic and growth effects found Picalm will be necessary to address whether both truncated later in development in the most phenotypically severe allele proteins are in fact stably produced in the mutant mice and, if (Picalmfit1–5R), however, do have their beginnings in the early they are, to assess the quantity and functional state of each fetus, in which defects in growth and hematopoiesis can be mutant protein. Overexpression of full-length human PICALM detected as early as day 14.5 of gestation (2). This suggests that or a C-terminal portion of the protein (residues 414–652) in cells at least some degree of AP2͞clathrin-mediated endocytosis must coimmunoprecipitates CHC and inhibits the uptake of trans- occur without Picalm, and that the endocytic processes involved ferrin and epidermal growth factor, whereas overexpression of in hematopoiesis, iron uptake͞metabolism, and growth are more the N-terminal 1–413-residue portion does not have these effects dependent on Picalm than those mediating other cellular func- (13). This indicates that the C-terminal segment contains the tions. The reports that overexpression of either PICALM (13) or major binding sites responsible for interacting with the endocytic AP180 (14) in transfected cells severely impairs the uptake of machinery both in vitro and in vivo. It is therefore apparent that, transferrin and epidermal growth factor by blocking coated-pit even if it is produced at an appreciable level, the small N- formation and endocytosis of the receptor–ligand complexes terminal portion of Picalm encoded by the mutant Picalmfit1–5R (14) are consistent with an important in vivo role for mouse allele (residues 1–116) will also not be able to interact with Picalm in the endocytic machinery involved in iron uptake and clathrin and the endocytic machinery and recruit them to the growth regulation. appropriate sites of coated-pit formation. Although the 1–413 The molecular architectures of AP180 and a related endocytic segment does not interact with CHC at a detectable level, the accessory protein, epsin 1, are designed for the rapid and 1–613 residue portion of human PICALM is capable of inter- efficient recruitment of clathrin and AP2 to sites of coated-pit acting with CHC but with less affinity than the complete protein formation in cellular membranes (27). Mutations in both lap and or the C-terminal 414–652 segment (13). This indicates that the

8364 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1432634100 Klebig et al. Downloaded by guest on September 28, 2021 1–569-residue portion encoded by the milder Picalmfit1–4R allele mutations generally result in defects in intracellular protein will at best interact with CHC with lower-than-normal affinity, sorting and the structure and function of lysosomes and storage which is consistent with the hypomorphic phenotype observed organelles such as melanosomes and platelet granules. The for this mutation in comparison to the more severe, and likely Picalmfit1 mutations may act independently, or, more likely, null, Picalmfit1–5R phenotype. implicate the involvement of clathrin͞AP2-mediated transport PICALM now joins the growing list of proteins involved in pathways in at least the developmental events observed as endocytosis and intracellular transport that, when mutated, lead abnormal in Picalmfit1 mutants (2). to a visible phenotype in mammals. Well-known examples are mutations in the AP3-mediated pathway, such as mocha (41, 42) ␦ ␤ We thank Dr. L. J. Hauser for help in the assembly of the BAC sequence; and pearl (43) mice (mutated and 3A subunits of the AP3 Drs. D. Johnson, Y. Liu, E. Michaud, and M. Garrick for suggestions on complex, respectively); Hermansky–Pudlak syndrome type-2 in the manuscript; and the Joint Genome Institute of the Department of humans (␤3A) (44); and pallid or muted mice, in which mutant Energy for sequencing the BAC. This research was sponsored by the pallidin or muted protein likely affects events later in vesicle- Office of Biological and Environmental Research, U.S. Department of trafficking such as vesicle-docking and fusion (45, 46). Such Energy, under Contract DE-AC05-00OR22725 with UT-Battelle, LLC.

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