Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics

Abstract

The structural maintenance of chromosomes (SMC) family member proteins previously were shown to play a critical role in mitotic chromosome condensation and segregation in yeast and Xenopus. Other family members were demonstrated to be required for DNA repair in yeast and mammals. Although several different SMC proteins were identified in different organisms, little is known about the SMC proteins in humans. Here, we report the identification of four human SMC proteins that form two distinct heterodimeric complexes in the cell, the human chromosome-associated protein (hCAP)-C and hCAP-E protein complex (hCAP-C/hCAP-E), and the human SMC1 (hSMC1) and hSMC3 protein complex (hSMC1/hSMC3). The hCAP-C/hCAP-E complex is the human ortholog of the Xenopus chromosome-associated protein (XCAP)-C/XCAP-E complex required for mitotic chromosome condensation. We found that a second complex, hSMC1/hSMC3, is required for metaphase progression in mitotic cells. Punctate vs. diffuse distribution patterns of the hCAP-C/hCAP-E and hSMC1/hSMC3 complexes in the interphase nucleus indicate independent behaviors of the two complexes during the cell cycle. These results suggest that two distinct classes of SMC protein complexes are involved in different aspects of mitotic chromosome organization in human cells.

Figure 1

Identification of three human SMC family proteins. (A) Sequence comparison of the conserved C-terminal domains of the SMC family proteins. Three PCR fragments C1, C2, and E1 obtained from human cDNAs are translated and compared with other family members [either SMC1(XCAP-C) or SMC2(XCAP-E) subfamily] in different species. Human SMC protein genes, to which the PCR fragments correspond, are indicated in parentheses. C1 (hCAP-C) and C2 (SB1.8/hSMC1) were aligned with XCAP-C (Xenopus) (3), cut3 (S. pombe) (10), Smc4 (S. cerevisiae data base), Smc1 (S. cerevisiae) (1), and hSMC1 (human SB1.8) (14). E1 (hCAP-E) was compared with XCAP-E (Xenopus) (3), scaffold protein II (chicken) (13), cut14 (S. pombe) (10), Smc2 (S. cerevisiae) (2), and Smc3 (S. cerevisiae database). Amino acids identical to C1 or E1 are shown as “⋅,” and three amino acids that distinguish two subfamilies are shown in bold letters (EKT vs. QRS). The numbers represent the amino acid positions of each protein. The amino acid sequences used to design PCR primers are underlined. (B) Sequence comparison of the N-terminal sequences of SMC proteins in vertebrates. The N-terminal sequence of BSMC1 (identical to the corresponding region of SB1.8) is compared with XCAP-C and hCAP-C (see amino acid numbers). The N-terminal sequence of hCAP-E is compared with those of XCAP-E, Scaffold protein II, and BSMC2 (15). Three amino acids exhibiting nonconserved changes in BSMC2 are indicated in boldfaced letters. (C) A schematic diagram of three human SMC protein cDNA clones. hSMC1 and hCAP-E cDNA clones are full-length, whereas the hCAP-C cDNA clone lacks the region corresponding to the first 80 amino acids. The conserved NTP-binding motif and DA box in the N and C termini are shown by ■ and ░⃞, respectively. The diverged coiled-coil domain in the middle is indicated. The thick underlines represent the region of proteins against which antibodies used in this study were raised.

Figure 2

Detection of the endogenous human SMC proteins. (A) Western blot analysis of the endogenous SMC proteins in a HeLa nuclear extract. HeLa crude nuclear extracts synchronized in S and M phases were applied to SDS/PAGE, transferred to a nitrocellulose membrane, and probed with the antibody indicated at the top. The sizes of the polypeptides detected are ≈165 kDa for hCAP-C (lane 1), 150 kDa for hSMC1 (lane 2), and 135 kDa for hCAP-E (lane 3). (B) Solubility of human SMC in the extract. Both nuclear extract (NE) and nuclear pellet (NP) (insoluble material left after the 0.4M salt extraction) were subjected to Western blot analysis, using the same antibodies as in A. (C) Silver staining of the heterodimeric human SMC complexes immunoprecipitated with antibodies specific for hCAP-C, hCAP-E or hSMC1 (lanes 1, 2, and 3, respectively). Immunoprecipitates were briefly washed with 1M guanidine-HCl. The polypeptide coprecipitated with hSMC1 is indicated as P140.

Figure 3

Immunofluorescent antibody staining analysis of hCAP-C/hCAP-E and hSMC1/P140 complexes in cells. (A) Localization of hCAP-C/hCAP-E complexes. DNA was detected by DAPI staining (panels 1 and 3). Immunofluorescent staining with anti-hCAP-E Ab of interphase and mitotic HeLa cells (panel 2) and chromosome spread of HT1080 cells (panel 4). (B) Localization of hSMC1/P140 complexes. Panels 1 and 3 are DAPI. Interphase (panel 2) and mitotic (panel 4) cells were stained with anti-hSMC1 Ab.

Figure 4

The effect of microinjection of anti-hSMC1 Ab in mitotic HeLa cells. Cells were fixed after 2.5 hr and the injected antibodies were detected by immunofluorescent staining with anti-rabbit IgG Ab (A) A cell injected during mid/late metaphase is arrested in mitosis. (Left) DAPI staining of DNA. (Right) Anti-(α)-rabbit IgG Ab staining the injected anti-hSMC1-Ab as indicated at the top. (B) A cell injected at early anaphase subsequently went through cytokinesis and resulted in two daughter cells. Again, DAPI staining and antibody staining are in left and right panels, respectively, as indicated. (C) Laser confocal microscopic analysis of cells injected with anti-hSMC1. Cells injected with antibody during mid/late metaphase (panel 1) and at early anaphase that subsequently underwent cytokinesis (panel 2).

Figure 5

A schematic diagram of the hSMC3 (HCAP) protein. The regions of protein corresponding to the peptide sequences obtained are indicated by the thick underlines. The conserved NTP-binding motif and DA box in the N and C termini are shown by ■ and ░⃞, respectively. The diverged coiled-coil domain in the middle is indicated.