Human telomere maintenance is essential for the protection of chromosome ends, and changes in telomere length have been implicated in ageing and cancer. Human telomere length is regulated by the TTAGGG-repeat-binding protein TRF1 and its interacting partners tankyrase 1, TIN2 and PINX1 (refs 5-9). As the TRF1 complex binds to the duplex DNA of the telomere, it is unclear how it can affect telomerase, which acts on the single-stranded 3' telomeric overhang. Here we show that the TRF1 complex interacts with a single-stranded telomeric DNA-binding protein--protection of telomeres 1 (POT1)--and that human POT1 controls telomerase-mediated telomere elongation. The presence of POT1 on telomeres was diminished when the amount of single-stranded DNA was reduced. Furthermore, POT1 binding was regulated by the TRF1 complex in response to telomere length. A mutant form of POT1 lacking the DNA-binding domain abrogated TRF1-mediated control of telomere length, and induced rapid and extensive telomere elongation. We propose that the interaction between the TRF1 complex and POT1 affects the loading of POT1 on the single-stranded telomeric DNA, thus transmitting information about telomere length to the telomere terminus, where telomerase is regulated.

Human telomere length is controlled by a negative feedback loop based on the binding of TRF1 to doublestranded telomeric DNA. The TRF1 complex recruits POT1, a single-stranded telomeric DNA-binding protein necessary for cis-inhibition of telomerase. By mass spectrometry, we have identified a new telomeric protein, which we have named POT1-interacting protein 1 (PIP1). PIP1 bound both POT1 and the TRF1-interacting factor TIN2 and could tether POT1 to the TRF1 complex. Reduction of PIP1 or POT1 levels with shRNAs led to telomere elongation, indicating that PIP1 contributes to telomere length control through recruitment of POT1.Supplemental material is available at http://www.genesdev.org.

Human telomeres contain two related telomeric DNAbinding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.The TTAGGG repeat arrays of mammalian telomeres associate with two related telomeric DNA-binding proteins, TRF1and TRF2 (1-3). These factors have closely related C-terminal Myb-type DNA binding domains and bind TTAGGG sequences as dimers or higher order oligomers. Dimerization is mediated by the TRF-homology domain, the signature motif of this family of telomeric proteins (4, 5). The crystal structure of the TRFH domains of TRF1 and TRF2 shows that the heterodimerization of TRF1 and TRF2 is impeded by crucial amino acid differences in the main dimerization interface (4, 5), and TRF1/ TRF2 heterodimers are not formed in vitro or in vivo (3). Therefore, the prevailing view has been that TRF1 and TRF2 form two separate complexes at telomeres.TRF1 recruits a number of other proteins to telomeres (reviewed in Ref. 6). The acidic N terminus of TRF1 binds to tankyrase 1 and 2 which are poly(ADP-ribose) polymerases that can modify TRF1 (7-11). ADP-ribosylation of TRF1 impedes its DNA binding activity in vitro, and tankyrase overexpression removes TRF1 from the telomeres and promotes its degradation. The in vitro ADP-ribosylation of TRF1 by tankyrase is inhibited by a second TRF1-interacting partner, TIN2 (12, 13). TIN2 appears to protect TRF1 from tankyrase in vivo, because RNAi 1 -mediated depletion of TIN2 results in tankyrase-dependent TRF1 loss (12, 13). TIN2 also functions to recruit PIP1 (also referred to as PTOP) to the TRF1 complex (14, 15). PIP1 is a POT1-interacting protein that med...

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATPbinding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6 -166p, is highly unstable. We show that its degradation involves the ubiquitin-proteasome system, as indicated by its in vivo stabilization in certain ubiquitin-proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6 -13p and Ste6 -90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.