©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Newly Synthesized Transferrin Receptors Can Be Detected in the Endosome before They Appear on the Cell Surface (*)

(Received for publication, January 24, 1995)

Clare E. Futter Christopher N. Connolly Daniel F. Cutler Colin R. Hopkins (§)

From the Medical Research Council Laboratory for Molecular Cell Biology and Department of Biology, University College London, Gordon Street, London WC1E 6BQ, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

It is well established that a proportion of newly synthesized lysosomal enzymes and class II major histocompatibility complex antigens are delivered directly to the endocytic pathway from the Golgi complex. Here we show that a significant proportion of newly synthesized transferrin receptors can be detected in endosomes before reaching the cell surface. These newly synthesized transferrin receptors are delivered to the endosome more efficiently than either constitutively secreted soluble proteins or glycophosphatidylinositol-anchored plasma membrane proteins suggesting that their transfer to the endosome is signal-dependent. Identification of a signal-dependent transfer step for proteins like the transferrin receptor operating on the exocytic pathway has important implications for membrane biogenesis, especially in the establishment of cell surface polarity.


INTRODUCTION

Newly synthesized proteins trafficking to the cell surface pass through the Golgi stack en route to a variety of destinations on the cell surface and inside the cell. Intracellular destinations include locations along regulated secretory pathways and endocytic routes. The earliest decisions in this selective routing are taken in trans-Golgi elements(1) , many of which contain clathrin coated domains whose ability to concentrate and selectively route subsets of trafficking proteins is well documented(2) . As yet, however, clathrin-coated domains are the only sorting mechanisms which have been definitively shown to be capable of selectively routing trafficking proteins in the TGN, (^1)and in view of the variety of alternative routes known to originate in this compartment it has become important to define more precisely the basis on which this selection is made. It seems likely, for example, that the first step in the sorting process is primarily concerned with selecting proteins carrying signals recognized by clathrin-coated domains so that further selective decisions are left to be made downstream, in the recycling pathways which receive these proteins(3) .

The best documented routes running directly from the trans-Golgi to intracellular destinations are those which carry mannose 6-phosphate receptors and major histocompatibility class II antigen-invariant chain complexes to the endocytic pathway(4) . These proteins are selected in the clathrin-coated domains of the trans-Golgi, and current evidence suggests that their recognition depends upon signals being carried in their cytoplasmic domains(4) . Here we determine if other newly synthesized membrane proteins which carry recognition signals for clathrin lattices in their cytoplasmic domains are also delivered to endosomal compartments directly from the Golgi.


EXPERIMENTAL PROCEDURES

Antibodies

Monoclonal antibodies B3/25 and H68.4 (to the extracellular and cytoplasmic domains, respectively) specific for the transferrin receptor were generously provided by I. Trowbridge (Salk Institute, San Diego). To prepare anti-Tfn-Rbulletgold complexes 10-nm gold particles were prepared and coupled to B3/25 as described previously(5) . Anti-alkaline phosphatase antibody was from Dakopatts Ltd (High Wycombe, United Kingdom). Rb9, a polyclonal antibody to the Tfn-R extracellular domain, was obtained from Ed Lennox (MRC Laboratory for Molecular Biology, Cambridge).

Cell Culture

HEp.2 (American Type Culture Collection CCL 23) cells were grown in Dulbecco's modified minimum essential medium (DMEM, Life Technologies Ltd., Paisley, Scotland) containing 10% FCS.

Generation of a Cell Line Stably Expressing Horseradish Peroxidase

A construct encoding horseradish peroxidase isoenzyme c was kindly provided by Amersham International plc (Amersham, UK). To enable entry into the secretory pathway, the signal sequence from human growth hormone(6) , kindly provided by H. J. Gilbert (University of Newcastle upon Tyne) was added. The construct was ligated into the pSRalpha plasmid (7) (a kind gift from DNAX, Palo Alto). A stable line of HEp.2 cells expressing this construct was produced by lipofection (8, 9, 10) .

Determination of the Rate of Traffic of Newly Synthesized Proteins to the Cell Surface

To determine the rate at which a soluble tracer of the constitutive secretory pathway is transferred from the Golgi to the cell surface cells stably expressing horseradish peroxidase were incubated for 4 h at 20 °C in DMEM containing 10% FCS and 20 mM HEPES. This treatment allows horseradish peroxidase activity to accumulate inside the cell. Cells were then transferred to 37 °C for various times in the presence of 100 µg/ml of cycloheximide to prevent further protein synthesis. Cells were then cooled to 4 °C and lysed in phosphate-buffered saline containing 1% Triton X-100. After removal of the nuclei by centrifugation, supernatants were assayed for horseradish peroxidase activity using o-phenylenediamine as substrate.

To determine the rate at which newly synthesized Tfn-R and alkaline phosphatase are transferred from the Golgi to the cell surface cells were pulsed with trans-S-label (150 µCi/ml) for 15 min in methionine-free medium and then chased in DMEM containing 10% FCS and 20 mM HEPES at 20 °C for 4 h. Cells were then transferred to 37 °C for various times before cooling to 4 °C. To detect Tfn-R at the cell surface, cells were then incubated in 100 µg/ml trypsin for 30 min at 4 °C(11) . The cleaved fragment of the extracellular domain of the Tfn-R and the intact receptor that remained cell associated were immunoprecipitated with Rb9. Immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by quantitation either by densitometry or using a PhosphorImager (Bio-Rad, UK).

To detect alkaline phosphatase at the cell surface, cells were incubated with 0.5 units/ml phosphatidylinositol-specific phospholipase C for 2 h at 4 °C. Alkaline phosphatase released and that remaining cell-associated was immunoprecipitated with anti-alkaline phosphatase antibody and analyzed as described for the Tfn-R.

Detection of Newly Synthesized Proteins in Endosomes

To determine whether horseradish peroxidase passes through endosomes en route to the cell surface, cells were incubated for 4 h at 20 °C in DMEM containing 10% FCS and 20 mM HEPES to accumulate horseradish peroxidase activity inside the cell. Anti-Tfn-Rbulletgold was added for the last hour at 20 °C. Cells were then transferred to 37 °C for various times in the presence of 100 µg/ml of cycloheximide and the continued presence of gold. Endosomes were then isolated as described (12) except that cells were used 24 h after plating.

Horseradish peroxidase activity in the endosome fraction and in an aliquot of the post-nuclear supernatant was measured after solubilization in 1% Triton X-100 and centrifugation at 100,000 times g for 1 h (to remove gold conjugates because they interfered with the horseradish peroxidase assay).

To determine whether newly synthesized Tfn-R and alkaline phosphatase pass through endosomes en route to the cell surface, cells were pulsed with trans-S-label (150 µCi/ml) for 15 min in methionine-free medium and then chased in DMEM containing 10% FCS and 20 mM HEPES at 20 °C for 4 h. Anti-Tfn-Rbulletgold was added for the last hour at 20 °C. Cells were then transferred to 37 °C for various times in the continued presence of anti-Tfn-Rbulletgold before cooling to 4 °C. Gold-loaded endosomes were then isolated as described above. Tfn-R and alkaline phosphatase were immunoprecipitated from an aliquot of the post-nuclear supernatant and from the endosome fraction. The proportion of newly synthesized Tfn-R and alkaline phosphatase in the endosome fraction was determined by quantitating the radioactivity in the immunoprecipitates as described above. To determine the proportion of total Tfn-R in the endosome fraction and hence the endosome yield, the immunoprecipitated Tfn-R were Western blotted with H68.4, and antibody binding was detected by chemiluminescence (ECL, Amersham, UK) and quantitated using a PhosphorImager.

Electron Microscopy

Cells stably expressing horseradish peroxidase were incubated for 4 h at 20 °C. Anti-Tfn-Rbulletgold was added for the last hour at 20 °C, and cells were then fixed. Fixation was carried out in Karnovsky fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 100 mM sodium cacodylate, pH 7.5(13) ). They were incubated with H(2)O(2) and diaminobenzidine prepared as described(14) , rinsed, osmicated, dehydrated, and embedded by standard procedures(5) . Sections were cut at either 70 nm, stained with lead citrate and uranyl acetate or cut 1 µm thick, stabilized with a thin film of evaporated carbon and viewed, unstained in a Phillips CM12 transmission electron microscope.


RESULTS

The Role of the Endosome Compartment in Constitutive Secretion

To obtain a marker of the constitutive secretory pathway, we have transfected mammalian cells with a cDNA encoding horseradish peroxidase linked to the signal sequence of human growth hormone, allowing the protein to gain access to the secretory pathway (10) . The enzyme is active and constitutively secreted in a number of different cell lines. In a HEp.2 cell line stably expressing horseradish peroxidase, the intracellular concentration of active enzyme is low. However, this can be increased by incubation of the cells at 20 °C, a temperature that inhibits movement out of the Golgi toward the cell surface but allows protein synthesis to continue (1) . Upon warming the cells to 37 °C in the presence of cycloheximide, the horseradish peroxidase activity accumulated in the Golgi is rapidly cleared from the cell with a half-time of approximately 20 min (Fig. 1).


Figure 1: Traffic of secretory horseradish peroxidase from the TGN to the cell surface. To measure the rate at which horseradish peroxidase is cleared from the cell HEp.2 cells stably expressing horseradish peroxidase were incubated at 20 °C for 4 h before transfer to 37 °C in the presence of cycloheximide. The percent horseradish peroxidase activity cleared from the cells was measured (squares). Results are means ± S.E. of three observations. To determine whether horseradish peroxidase passes through endosomes en route to the cell surface cells were incubated at 20 °C for 4 h and anti-Tfn-Rbulletgold was added for the last hour at 20 °C. Cells were then transferred to 37 °C in the presence of cycloheximide and the continued presence of anti-Tfn-Rbulletgold. Gold-loaded endosomes were isolated and the horseradish peroxidase activity recovered in the endosome fractions as a percent of that in the post-nuclear supernatant was measured (circles). Results are means ± S.E. of three observations.



To determine whether a significant proportion of horseradish peroxidase passes through endosomes en route to the cell surface, cells expressing horseradish peroxidase were incubated for 4 h at 20 °C and during the last hour of incubation at 20 °C anti-Tfn-Rbulletgold was added. Cells were then transferred to 37 °C in the presence of cycloheximide and anti-Tfn-Rbulletgold-loaded endosomes isolated as described previously(12) . Morphological analysis shows that the entire Tfn recycling pathway is accessible at 20 °C, and neither the yield nor the form of the endosomes changes significantly at this reduced temperature. As shown in Fig. 1less than 5% of the post-nuclear supernatant horseradish peroxidase activity was recovered in the anti-Tfn-Rbulletgold-isolated endosomes at any chase time studied (Fig. 1). Cells incubated as above were also examined by electron microscopy. In conventional thin sections the distribution of anti-Tfn-Rbulletgold at the end of the 20 °C incubation is seen in large (0.2-0.5 µm diameter) vacuoles and smaller (50-80 nm diameter) vesicles but is not found in the flattened cisternae of the Golgi stack or their associated vacuoles labeled with horseradish peroxidase (Fig. 2). Sections up to 1 µm thick were also examined to determine whether there were continuities between structures labeled with horseradish peroxidase and those containing anti-Tfn-Rbulletgold which would not be evident in thin sections. As shown in Fig. 3, although components containing the two tracers are closely associated double-labeled elements were not observed. We conclude, therefore, that TGN components are unlikely to be present in the anti-Tfn-Rbulletgold-loaded fractions. In a previously published study (10) , we showed that if accumulation of horseradish peroxidase activity in Golgi elements at 20 °C is followed by transfer to 37 °C, the horseradish peroxidase and anti-Tfn-Rbulletgold tracers remain distributed in separate elements.


Figure 2: Thin section electron microscopy of the intracellular distribution of secretory horseradish peroxidase and the transferrin receptor. Conventional thin section (70 nm thick) of HEp.2 cells stably expressing horseradish peroxidase incubated for 3 h at 20 °C and then incubated for 1 h at 20 °C with anti-Tfn-Rbulletgold. Reaction product for horseradish peroxidase is contained within Golgi cisternae and their associated vesicles. The anti-Tfn-Rbulletgold labels large vacuolar endosomes and small vesicles (arrowed) in the vicinity. Bar, 0.2 µm.




Figure 3: Thick section electron microscopy of the intracellular distribution of secretory horseradish peroxidase and the transferrin receptor. Thick sections (1.0 µm) of HEp.2 cells stably expressing horseradish peroxidase and incubated as in Fig. 2. Thick sections demonstrate the extensive continuity which exists between Golgi complex elements and show how closely associated they can be with endocytic elements containing gold tracer. However, even in sections which are up to 10 times thicker than the diameter of the 70-120 nm diameter vesicles and tubules the tracers are seen to be distributed in separate elements. Large arrows point to Golgi stack; arrowheads indicate gold-labeled vesicles; MVE, multivesicular endosomes; C, centriole. Bars, 0.2 µm.



Transfer of Newly Synthesized Tfn-R Directly from the Golgi to the Endosome

To determine the rate at which newly synthesized proteins reached gold-loaded endosomes, cells were pulsed for 15 min at 37 °C with trans-S-label and chased at 20 °C for 4 h with anti-Tfn-Rbulletgold being added for the last hour of incubation. Cells were then transferred to 37 °C, endosomes isolated as described under ``Experimental Procedures,'' and radiolabeled Tfn-R and alkaline phosphatase were immunoprecipitated.

Compared to the content of pre-existing Tfn-R in the endosome fraction the content of newly synthesized Tfn-R in the endosomes isolated from 20 °C incubated cells is low (Fig. 4a). This is consistent with the expectation that most of the newly synthesized Tfn-R will be retained in the Golgi at this temperature. On transfer to 37 °C, there is a rapid increase in the content of newly synthesized Tfn-R in the endosome fraction. This increase, which reaches a peak in the first 10 min is then followed by a decline over the next 10 min. The amount of newly synthesized alkaline phosphatase in endosome fractions from cells incubated at 20 °C is low and does not show any detectable increase when cells are shifted to 37 °C.


Figure 4: Kinetics of appearance of newly synthesized protein in endosomes and on the plasma membrane. a, to measure the rate of appearance of newly synthesized Tfn-R in endosomes HEp.2 cells were pulse labeled with trans-S-label for 15 min and chased for 4 h at 20 °C. Anti-Tfn-Rbulletgold was added for the last hour at 20 °C before transfer to 37 °C in the continued presence of anti-Tfn-R/gold. Gold-loaded endosomes were then isolated. The amount of newly synthesized Tfn-R (squares) and alkaline phosphatase (circles) in the endosome fraction was measured by immunoprecipitation and the total amount of Tfn-R (triangles) in the endosome fraction was measured by Western blotting. Results are those of a single experiment where measurement of all these parameters was performed on the same cells. b, to measure the rate of appearance of newly synthesized Tfn-R at the cell surface HEp.2 cells were pulse labeled with trans-S-label for 15 min at 37 °C and then chased for 4 h at 20 °C before transfer to 37 °C. The amount of Tfn-R at the cell surface was determined by measuring the proportion of newly synthesized Tfn-R accessible to cleavage with trypsin at 4 °C (triangles). Results are means ± S.E. of three observations. The rate of appearance of newly synthesized Tfn-R (squares) and alkaline phosphatase (circles) in endosomes was measured as described in Fig. 4a. Results are means ± S.E. of four observations.



To determine whether newly synthesized Tfn-R reach the endosome before they can be detected on the cell surface, the rate at which newly synthesized Tfn-R and alkaline phosphatase are transported from the Golgi to the plasma membrane was measured. Cells were pulsed for 15 min at 37 °C with trans-S-label and incubated in chase medium at 20 °C for 4 h. Cells were then transferred to 37 °C. The amount of newly synthesized Tfn-R appearing on the cell surface was determined by incubating the cells with trypsin at 4 °C. Under these conditions the extracellular domain of the Tfn-R is cleaved and can be immunoprecipitated with more than 80% efficiency(5) . The amount of newly synthesized alkaline phosphatase appearing on the cell surface was determined by incubating cells with phosphatidylinositol-specific phospholipase C at 4 °C. This enzyme cleaves the labeled alkaline phosphatase which was then immunoprecipitated.

As shown in Fig. 4b, there is a steady increase in the amount of newly synthesized Tfn-R on the cell surface from 10 min at 37 °C when less than 5% can be detected to 30 min when, as shown by more extended incubations, a steady state of 30% is reached. Thus the rise in the amount of newly synthesized Tfn-R in endosomes at 10 min occurs before significant amounts of newly synthesized Tfn-R can be detected at the cell surface. It is noteworthy that the decline in the amount of newly synthesized Tfn-R in endosomes that occurs at 20 min is coincident with the rise in newly synthesized Tfn-R at the cell surface. Although, as shown in Fig. 4b, a small amount (6%) of newly synthesized Tfn-R is found in the endosome at the end of the 20 °C incubation, there is no indication that a similar leakage occurs to the cell surface at this temperature.

Newly synthesized alkaline phosphatase reaches the cell surface with similar kinetics to newly synthesized Tfn-R (results not shown).


DISCUSSION

A previous cell fractionation study on the hepatocyte cell line HepG2 by Stoorvogel et al.(15) found a significant colocalization of a secretory protein with an endocytosed Tfn-horseradish peroxidase conjugate, and their correlative morphological analysis suggested this was due to the presence of endocytosed Tfn tracer in the trans-Golgi reticulum. In the present study on HEp.2, an epithelioid cell line, we have shown that the anti-Tfn-Rbulletgold tracer used to load the endosomes does not enter the TGN. This is apparent both from the cell fractionation work, which shows that fractions containing endocytosed anti-Tfn-Rbulletgold contain very low levels of horseradish peroxidase after a 20 °C block, and from the electron microscopy, which shows horseradish peroxidase accumulates in the TGN at 20 °C but remains separate from gold-loaded endosomes. Endosomes loaded with anti-Tfn-Rbulletgold contain extremely low levels of newly synthesized alkaline phosphatase as well as horseradish peroxidase at 20 °C, and no increase is seen on transfer to 37 °C when the newly synthesized proteins transfer to the cell surface. We conclude, therefore, that in HEp.2 cells routes for constitutive secretion do not pass through gold-loaded endosomes. However, since our previous studies (12) showed by electron microscopy that endosome fractions obtained by this gold-mediated density shift protocol are composed primarily of the vacuolar elements of the Tfn-R recycling pathway we cannot exclude the possibility that these soluble phase and GPI-anchored proteins may be transferred to the cell surface via a later step in the recycling pathway. They could for example be transferred via the 60-nm tubules in the pericentriolar area which form the most distal compartment in the Tfn-R recycling pathway in these cells(16) , since these tubules are not well represented in our density shifted fractions(12) . The demonstration that horseradish peroxidase expressed from transfected cDNA is efficiently cleared from the cell supports the view that this fluid-phase tracer moves directly to the surface from the TGN because a fluid-phase tracer entering the vacuolar elements of the endocytic pathway would be more likely to move to the lysosome than recycle to the cell surface(3) .

Our data demonstrate that the endosome content of newly synthesized Tfn-R increases approximately 3-fold when the cells are shifted from 20 to 37 °C. No increases were seen with either horseradish peroxidase or alkaline phosphatase which provide rigorous internal controls for the isolation procedure. The content of newly synthesized Tfn-R within the purified endosome fraction peaks 10 min after temperature shift at 15% of the total radiolabeled Tfn-R. It is important to emphasize that this percentage is a considerable underestimate of the endosome content because the purification procedure recovers only about 30% of the endosomal Tfn-R. This estimate is based on the percentage of total Tfn-R in the endosome fraction (approximately 20%), the earlier demonstration that the endosome fraction provides a 150-200-fold purification containing less than 1% plasma membrane(12) , and the percentage (70%) of total Tfn-R that are intracellular (assayed by stripping cells incubated to steady state with I-Tfn with low pH at 4 °C). Correcting for the loss of endosome elements during fractionation suggests that the endosome compartment as a whole could contain more than three times (70/20) the amount of newly synthesized Tfn-R we detect in purified fractions.

The efficiency with which newly synthesized Tfn-R are delivered to gold-loaded endosomes compared to fluid-phase horseradish peroxidase and GPI-linked alkaline phosphatase suggests that it is likely to be a signal dependent process. The only known trafficking signal carried by Tfn-R is the well characterized tyrosine-based internalization motif which is recognized by the selection mechanism of the clathrin-coated pits on the plasma membrane(3) . There is no evidence that this motif is recognized by the sorting mechanisms located within the clathrin lattices of the TGN, although other signals such as the dileucine signal of the CD3y subunit of the T cell antigen receptor(17) , and the signals of the invariant chain (18) which are recognized in the TGN are also known to be recognized by the clathrin lattices of the plasma membrane. It would not be very surprising therefore to find that the internalization signal carried by the Tfn-R is recognized in the TGN so that this protein like the CD3y chain and invariant chain are then sorted directly into the endosome compartment.

The suggestion that the internalization signal in the cytoplasmic domain of a predominantly cell surface protein like the Tfn-R is also recognized by clathrin lattices in the trans-Golgi has important implications for trafficking in polarized cells since it implies proteins carrying a plasma membrane internalization signal and routed through the TGN could be routed through the endosome. If this is so, then a means of ensuring that proteins trafficking through the endosome are transferred to the appropriate surface domain would also be required. In the proteins studied thus far this requirement has been satisfied. Thus in the polymeric IgA receptor which has a very efficient plasma membrane-coated pit signal there is also a signal which prevents transfer from the endosome to the apical surface(19) . Similarly in the low density lipoprotein receptor there is a proximal basolateral targetting signal which overlaps with the well characterized plasma membrane coated pit signal, as well as a distal signal, independent of the internalization signal, which prevents apical transfer(20, 21) . Interestingly, removal of the segment of the cytoplasmic domain of Tfn-R which includes the tyrosine-containing internalization signal is reported to have no influence on its delivery to the basolateral surface(22) . However, while the YTRF sequence in the Tfn-R has been shown to be all that is required for internalization in coated pits(23) , site-directed mutagenesis has shown that other regions of the cytoplasmic domain can also promote coated pit-mediated uptake at the plasma membrane(24) .

The proposal that closely related or identical signals may be recognized with varying affinity by clathrin-coated domains in both the plasma membrane and the TGN(3) , together with the knowledge that interaction with clathrin-coated domains at both sites results in delivery to the endosome, gives the endosome a central role in the selective routing of membrane proteins. It may be that the initial site of sorting on the biosynthetic pathway is the TGN and that it is the mechanisms for sorting which reside within the endosome which are primarily responsible for the delivery of proteins to the plasma membrane, the lysosome, the trans-Golgi and other intracellular destinations, such as the synaptic vesicle(25, 26) . The number and nature of the sorting mechanisms within the endosome responsible for these decisions has yet to be determined, but they probably rely upon interactions other than those which operate within clathrin-coated domains.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: MRC Laboratory for Molecular Cell Biology, University College London, Gordon St., London WC1E 6BQ, United Kingdom. Tel.: 071-380-7806; Fax: 071-380-7805.

(^1)
The abbreviations used are: TGN, trans-Golgi network; ALP, alk aline phosphatase; GPI, glycophosphatidylinositol; Tfn-R, transferrin receptor; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.


REFERENCES

  1. Griffiths, G., and Simons, K. (1986) Science 234, 438-443 [Medline] [Order article via Infotrieve]
  2. Pearse, B. M. F., and Robinson, M. S. (1990) Annu. Rev. Cell Biol. 6, 151-171 [CrossRef]
  3. Trowbridge, I. S., Collawn, J. F., and Hopkins, C. R. (1994) Annu. Rev. Cell Biol. 9, 129-162 [CrossRef]
  4. Sandoval, I. V., and Bakke, O. (1994) Trends Cell Biol. 4, 292-297 [CrossRef]
  5. Hopkins, C. R., and Trowbridge, I. S. (1983) J. Cell Biol. 97, 508-521 [Abstract]
  6. Hall, J., Hazlewood, G. P., Surani, M. A., Hirst, B. H., and Gilbert, H. J. (1990) J. Biol. Chem. 265, 19996-19999 [Abstract/Free Full Text]
  7. Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K., Yoshida, M., and Arai, N. (1988) Mol. Cell. Biol. 8, 466-472 [Medline] [Order article via Infotrieve]
  8. Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7413-7417 [Abstract]
  9. Chang, A. C. Y., and Brenner, D. G. (1988) Focus 10, 66-69
  10. Connolly, C. N., Futter, C. E., Gibson, A., Hopkins, C. R., and Cutler, D. F. (1994) J. Cell Biol. 127, 641-652 [Abstract]
  11. Trowbridge, I. S., and Omary, M. B. (1981) Proc. Natl. Acad. Sci.U. S. A. 78, 3039-3043 [Abstract]
  12. Futter, C. E., and Hopkins, C. R. (1989) J. Cell Sci. 94, 685-694 [Abstract]
  13. Karnovsky, M. J. (1965) J. Cell Biol. 27, 137a
  14. Graham, R. C., and Karnovsky, M. J. (1966) J. Histochem. Cytochem. 14, 291-302 [Medline] [Order article via Infotrieve]
  15. Stoorvogel, W., Geuze, H. J., Griffith, J. M., and Strous, G. J. (1988) J. Cell Biol. 106, 1821-1829 [Abstract]
  16. Hopkins, C. R., Gibson, A., Shipman, M., Strickland, D., and Trowbridge, I. S. (1994) J. Cell Biol. 125, 1265-1274 [Abstract]
  17. Letourner, F., and Klausner, R. D. (1992) Cell 69, 1143-1157 [Medline] [Order article via Infotrieve]
  18. Odorizzi, C. G., Trowbridge, I. S., Xue, L., Hopkins, C. R., Davis, C. D., and Collawn, J. F. (1994) J. Cell Biol. 126, 317-330 [Abstract]
  19. Casanova, J. E., Apodaca, G., and Mostov, K. E. (1991) Cell 66, 65-75 [Medline] [Order article via Infotrieve]
  20. Matter, K., Hunziker, W., and Mellman, I. (1992) Cell 71, 741-753 [Medline] [Order article via Infotrieve]
  21. Matter, K., Whitney, J. A., Yamamoto, E. M., and Mellman, I. (1993) Cell 74, 1053-1064 [Medline] [Order article via Infotrieve]
  22. Dargemont, C., LeBivic, A., Rothenburger, S., Iacopetta, B., and Kuhn, L. (1993) EMBO J. 12, 1713-1721 [Abstract]
  23. Collawn, J. F., Stangel, M., Kuhn, L. A., Esekogwu, V., Jing, S., Trowbridge, I. S., and Tainer, J. A. (1990) Cell 63, 1061-1072 [Medline] [Order article via Infotrieve]
  24. McGraw, T. E., Pytowski, B., Arzt, J., and Ferrone, C. (1991) J. Cell Biol. 112, 853-861 [Abstract]
  25. Bauerfeind, R., Regnier-Vigouroux, A., Flatmark, T., and Huttner, W. B. (1993) Neuron 11, 105-121 [Medline] [Order article via Infotrieve]
  26. Regnier-Vigouroux, A., Tooze, S. A., and Huttner, W. B. (1991) EMBO J. 10, 3589-3601 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.