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© The Rockefeller University Press, 0021-9525/1998//319 $5.00
The Journal of Cell Biology, Volume 143, Number 2, , 1998 319-331

Regular Articles

The Membrane Transport Factor TAP/p115 Cycles between the Golgi and Earlier Secretory Compartments and Contains Distinct Domains Required for Its Localization and Function



David S. Nelson*,{ddagger}, Cecilia Alvarez*, Ya-sheng Gao*, Rafael García-Mata*, Elizabeth Fialkowski*, and Elizabeth Sztul*

* Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294; and {ddagger} Department of Molecular Biology, Princeton University, Princeton, New Jersey 08543

The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al., 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15°C treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi.

TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH2-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH2-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.

Key Words: TAP • p115 • Golgi • GM 130 • VTC

Abbreviations used in this paper: DOC, deoxycholate; ERGIC, ER to Golgi intermediate compartment; IF, immunofluorescence; NSF, N-ethylmaleimide–sensitive factor; PNS, postnuclear supernatant; SG, stacked Golgi; SNAP, soluble NSF atttachment protein; SNARE, N-ethylmaleimide–sensitive factor attachment protein; t-SNARE, target SNARE; v-SNARE, vesicle SNARE; VTC, vesicular tubular cluster.

D.S. Nelson and C. Alvarez contributed equally to this work.

Address all correspondence to E. Sztul, Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294. Tel.: (205) 934-1465. Fax: (205) 975-9131. E-mail: esztul{at}bmg.bhs.uab.edu

D.S. Nelson's present address is Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294.


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